Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells

ABSTRACT

The invention concerns a new urate oxidase activity protein which has the following sequence: 
     
         ______________________________________                                    
 
    
          Ser    Ala    Val  Lys  Ala  Ala  Arg  Tyr  Gly                      
Lys  Asp    Asn    Val  Arg  Val  Tyr  Lys  Val  His                      
Lys  Asp    Glu    Lys  Thr  Gly  Val  Gln  Thr  Val                      
Tyr  Glu    Met    Thr  Val  Cys  Val  Leu  Leu  Glu                      
Gly  Glu    Ile    Glu  Thr  Ser  Tyr  Thr  Lys  Ala                      
Asp  Asn    Ser    Val  Ile  Val  Ala  Thr  Asp  Ser                      
Ile  Lys    Asn    Thr  Ile  Tyr  Ile  Thr  Ala  Lys                      
Gln  Asn    Pro    Val  Thr  Pro  Pro  Glu  Leu  Phe                      
Gly  Ser    Ile    Leu  Gly  Thr  His  Phe  Ile  Glu                      
Lys  Tyr    Asn    His  Ile  His  Ala  Ala  His  Val                      
Asn  Ile    Val    Cys  His  Arg  Trp  Thr  Arg  Met                      
Asp  Ile    Asp    Gly  Lys  Pro  His  Pro  His  Ser                      
Phe  Ile    Arg    Asp  Ser  Glu  Glu  Lys  Arg  Asn                      
Val  Gln    Val    Asp  Val  Val  Glu  Gly  Lys  Gly                      
Ile  Asp    Ile    Lys  Ser  Ser  Leu  Ser  Gly  Leu                      
Thr  Val    Leu    Lys  Ser  Thr  Asn  Ser  Gln  Phe                      
Trp  Gly    Phe    Leu  Arg  Asp  Glu  Tyr  Thr  Thr                      
Leu  Lys    Glu    Thr  Trp  Asp  Arg  Ile  Leu  Ser                      
Thr  Asp    Val    Asp  Ala  Thr  Trp  Gln  Trp  Lys                      
Asn  Phe    Ser    Gly  Leu  Gln  Glu  Val  Arg  Ser                      
His  Val    Pro    Lys  Phe  Asp  Ala  Thr  Trp  Ala                      
Thr  Ala    Arg    Glu  Val  Thr  Leu  Lys  Thr  Phe                      
Ala  Glu    Asp    Asn  Ser  Ala  Ser  Val  Gln  Ala                      
Thr  Met    Tyr    Lys  Met  Ala  Glu  Gln  Ile  Leu                      
Ala  Arg    Gln    Gln  Leu  Ile  Glu  Thr  Val  Glu                      
Tyr  Ser    Leu    Pro  Asn  Lys  His  Tyr  Phe  Glu                      
Ile  Asp    Leu    Ser  Trp  His  Lys  Gly  Leu  Gln                      
Asn  Thr    Gly    Lys  Asn  Ala  Glu  Val  Phe  Ala                      
Pro  Gln    Ser    Asp  Pro  Asn  Gly  Leu  Ile  Lys                      
Cys  Thr    Val    Gly  Arg  Ser  Ser  Leu  Lys  Ser                      
Lys  Leu                                                                  
______________________________________                                    
 
     optionally preceeded by a methionine, or in that it may present a degree of substantial homology with this sequence. 
     The invention is also aimed at medicines containing this protein as well as the genetic engineering implements to obtain it.

This application is a continuation of application Ser. No. 07/659,408, filed Apr. 25, 1991, now abandoned.

The invention relates to a novel protein possessing urate oxidase activity; the invention also concerns the drugs containing this protein as well as the genetic engineering tools for producing that protein and notably the recombinant gene coding for that protein, the expression vector carrying that gene and the eukaryotic cells or the prokaryotic microorganisms transformed by this expression vector.

Urate oxidase (EC 1.7.3.3.), which is also called uricase, is an enzyme of the purine degradation pathway. This enzyme does not exist in primates (such as man), birds, a few reptiles or most insects. It is also non-existent in some dogs (such as the dalmatian).

In man, the purine bases--adenine and guanine are--converted to xanthine. The xanthine is oxidized by xanthine oxidase to form uric acid according to the following reaction:

    xanthine+H.sub.2 O+O.sub.2 →uric acid+O.sub.2 -

The O₂ - radical, which is the substrate for superoxide dismutase, is converted by the latter to hydrogen peroxide.

Uric acid, a metabolite present in blood, is normally found essentially in the form of the soluble monosodium salt. However, in certain people, it may happen that the uric acid precipitates and forms calculi. Hyperuricemia, which is an increase in the amount of uric acid circulating in the blood, causes uric acid to deposit in the cartilaginous tissues, leading to gout. Hyperuricemia can also have consequences on the kidneys: an excess of uric acid in the urine and in the kidneys can result in uric acid nephrolithiasis, i.e. the accumulation of renal calculi, which are very painful and can damage the kidney. These calculi are composed of uric acid possibly associated with phosphate and oxalate salts. Overproduction of uric acid can have a variety of origins: congenital metabolic defects, Lesch-Nyhan syndrome, excess ingestion of purine or proteins, treatments with uricosuric drugs, treatments of the hemopathies, particularly the cancerous hemopathies by cytolytic agents (chemotherapy) or by radiotherapy. (Gutman, A. B. and YU, T. F. (1968) Am. J. Med. 45-756-779).

Urate oxidase, the enzyme which catalyzes the degradation of uric acid to allantoin (a compound which is much more soluble than uric acid and does not crystallize at the concentrations reached in biological fluids), therefore has therapeutic value. Used in injections, it has a large number of advantages in the treatment of hyperuricemia and nephrolithiasis: speed of the hypouricemic effect (reduction of hyperuricemia of the order of 50% in less than 24 h), better protection of the kidney against lithiasis compared with other drugs such as allopurinol (a xanthine oxidase inhibitor), etc. At the present time, this enzyme is mainly used as adjuvant for the cytolytic agents in chemotherapy.

The urate oxidase currently used as a drug is obtained by a method comprising the culture of a mycelium of Aspergillus flavus and isolation of the urate oxidase from the culture medium by extraction, together with several steps for purifying this protein. This method, which makes it possible to obtain urate oxidase of high purity, nevertheless has disadvantages. In fact, the physiology and especially the genetics of A. flavus are not easy to work with (WOLOSHUK et al. (1989) Applied environ. microbiol., vol. 55, p. 86-90). It is therefore impossible to obtain strains which produce this enzyme in substantial amounts. Furthermore, A. flavus is liable to produce aflatoxins, which are sometimes difficult to separate off. The purified product should consequently be checked to ensure that it is free from these toxins.

There is therefore a need for a purer urate oxidase of A. flavus as well as for genetic engineering tools and techniques whereby these disadvantages can be overcome.

The Applicant purified the urate oxidase extracted from A. flavus, named thereafter the urate oxidase extract, up to a purity degree higher than that already known for this protein; the Applicant also determined the partial sequence of that protein and built two pools of labelled probes able to hybridize with the nucleotides coding for two portions of that protein. It then constructed an expression vector comprising this cDNA, transformed a strain of E. coli K12 with the latter, cultivated said strain and verified that the lyzate of the cells contained a recombinant protein of the expected molecular weight, which possesses urate oxidase activity (capacity to degrade uric acid allantoine).

The Applicant also constructed several vectors for expression in eukaryotic cells, comprising a recombinant gene coding for urate oxidase whose sequence contains variations, relative to the isolated cDNA, introduced for the purpose of inserting codons which are customary in eukaryotic cells, transformed different eukaryotic cells with the aid of these vectors, cultivated said cells in a small volume as well as in a larger volume (fermenter), and found that the lyzates of the cells contained a substantial proportion of a recombinant protein of the expected molecular weight, possessing urate oxidase activity. It purified this recombinant protein and partially characterized it, comparatively towards the urate oxidase extract.

Therefore, the present invention relates to a novel protein possessing a specific urate oxidase activity of at least 16 U/mg, which has the following sequence (SEQUENCE ID NO. 1):

    ______________________________________                                         Ser  Ala    Val    Lys  Ala  Ala  Arg  Tyr  Gly  Lys                           Asp  Asn    Val    Arg  Val  Tyr  Lys  Val  His  Lys                           Asp  Glu    Lys    Thr  Gly  Val  Gln  Thr  Val  Tyr                           Glu  Met    Thr    Val  Cys  Val  Leu  Leu  Glu  Gly                           Glu  Ile    Glu    Thr  Ser  Tyr  Thr  Lys  Ala  Asp                           Asn  Ser    Val    Ile  Val  Ala  Thr  Asp  Ser  Ile                           Lys  Asn    Thr    Ile  Tyr  Ile  Thr  Ala  Lys  Gln                           Asn  Pro    Val    Thr  Pro  Pro  Glu  Leu  Phe  Gly                           Ser  Ile    Leu    Gly  Thr  His  Phe  Ile  Glu  Lys                           Tyr  Asn    His    Ile  His  Ala  Ala  His  Val  Asn                           Ile  Val    Cys    His  Arg  Trp  Thr  Arg  Met  Asp                           Ile  Asp    Gly    Lys  Pro  His  Pro  His  Ser  Phe                           Ile  Arg    Asp    Ser  Glu  Glu  Lys  Arg  Asn  Val                           Gln  Val    Asp    Val  Val  Glu  Gly  Lys  Gly  Ile                           Asp  Ile    Lys    Ser  Ser  Leu  Ser  Gly  Leu  Thr                           Val  Leu    Lys    Ser  Thr  Asn  Ser  Gln  Phe  Trp                           Gly  Phe    Leu    Arg  Asp  Glu  Tyr  Thr  Thr  Leu                           Lys  Glu    Thr    Trp  Asp  Arg  Ile  Leu  Ser  Thr                           Asp  Val    Asp    Ala  Thr  Trp  Gln  Trp  Lys  Asn                           Phe  Ser    Gly    Leu  Gln  Glu  Val  Arg  Ser  His                           Val  Pro    Lys    Phe  Asp  Ala  Thr  Trp  Ala  Thr                           Ala  Arg    Glu    Val  Thr  Leu  Lys  Thr  Phe  Ala                           Glu  Asp    Asn    Ser  Ala  Ser  Val  Gln  Ala  Thr                           Met  Tyr    Lys    Met  Ala  Glu  Gln  Ile  Leu  Ala                           Arg  Gln    Gln    Leu  Ile  Glu  Thr  Val  Glu  Tyr                           Ser  Leu    Pro    Asn  Lys  His  Tyr  Phe  Glu  Ile                           Asp  Leu    Ser    Trp  His  Lys  Gly  Leu  Gln  Asn                           Thr  Gly    Lys    Asn  Ala  Glu  Val  Phe  Ala  Pro                           Gln  Ser    Asp    Pro  Asn  Gly  Leu  Ile  Lys  Cys                           Thr  Val    Gly    Arg  Ser  Ser  Leu  Lys  Ser  Lys                           Leu,                                                                           ______________________________________                                    

optionally preceded by a methionine or which present a substantial degree of homology with that sequence.

Preferably the specific urate oxidase activity of the invention protein is of about 30 U/mg.

A preferred protein of that type is the protein, which, by analysis on a bidimensional gel, presents a spot of molecular mass of about 33.5 kDa and an isoelectric point around 8.0, representing at least 90% of the protein mass.

Preferably the purity degree of the invention protein, determined by liquid chromatography on aC8 grafted silica column, is higher than 80%.

An interesting protein of that type is the protein having an isoelectric point of 8.0. Preferably the amino-terminal serine of that protein carries a blocking group, having preferably a mass around 43 units of atomic mass, such as for example the acetyl group.

The present invention also relates to the drug which contains the invention protein in combination with a pharmaceutically acceptable carrier. The invention protein may advantageously replace, in its different uses, the urate oxidase extract possessing a specific urate oxidase activity of about 8 U/mg, which is sold in the injectable form under the trade mark "Uricozyme" (Vidal 1990).

The invention also relates to a recombinant gene which comprises a DNA sequence coding for the protein having the following sequence (SEQUENCE ID NO. 2):

    __________________________________________________________________________     Met                                                                               Ser                                                                               Ala                                                                               Val                                                                               Lys                                                                               Ala                                                                               Ala                                                                               Arg                                                                               Tyr                                                                               Gly                                                                               Lys                                                                               Asp                                                                               Asn                                                                               Val                                                                               Arg                                                                               Val                                                                               Tyr                            Lys                                                                               Val                                                                               His                                                                               Lys                                                                               Asp                                                                               Glu                                                                               Lys                                                                               Thr                                                                               Gly                                                                               Val                                                                               Gln                                                                               Thr                                                                               Val                                                                               Tyr                                                                               Glu                                                                               Met                                                                               Thr                            Val                                                                               Cys                                                                               Val                                                                               Leu                                                                               Leu                                                                               Glu                                                                               Gly                                                                               Glu                                                                               Ile                                                                               Glu                                                                               Thr                                                                               Ser                                                                               Tyr                                                                               Thr                                                                               Lys                                                                               Ala                                                                               Asp                            Asn                                                                               Ser                                                                               Val                                                                               Ile                                                                               Val                                                                               Ala                                                                               Thr                                                                               Asp                                                                               Ser                                                                               Ile                                                                               Lys                                                                               Asn                                                                               Thr                                                                               Ile                                                                               Tyr                                                                               Ile                                                                               Thr                            Ala                                                                               Lys                                                                               Gln                                                                               Asn                                                                               Pro                                                                               Val                                                                               Thr                                                                               Pro                                                                               Pro                                                                               Glu                                                                               Leu                                                                               Phe                                                                               Gly                                                                               Ser                                                                               Ile                                                                               Leu                                                                               Gly                            Thr                                                                               His                                                                               Phe                                                                               Ile                                                                               Glu                                                                               Lys                                                                               Tyr                                                                               Asn                                                                               His                                                                               Ile                                                                               His                                                                               Ala                                                                               Ala                                                                               His                                                                               Val                                                                               Asn                                                                               Ile                            Val                                                                               Cys                                                                               His                                                                               Arg                                                                               Trp                                                                               Thr                                                                               Arg                                                                               Met                                                                               Asp                                                                               Ile                                                                               Asp                                                                               Gly                                                                               Lys                                                                               Pro                                                                               His                                                                               Pro                                                                               His                            Ser                                                                               Phe                                                                               Ile                                                                               Arg                                                                               Asp                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Arg                                                                               Asn                                                                               Val                                                                               Gln                                                                               Val                                                                               Asp                                                                               Val                                                                               Val                            Glu                                                                               Gly                                                                               Lys                                                                               Gly                                                                               Ile                                                                               Asp                                                                               Ile                                                                               Lys                                                                               Ser                                                                               Ser                                                                               Leu                                                                               Ser                                                                               Gly                                                                               Leu                                                                               Thr                                                                               Val                                                                               Leu                            Lys                                                                               Ser                                                                               Thr                                                                               Asn                                                                               Ser                                                                               Gln                                                                               Phe                                                                               Trp                                                                               Gly                                                                               Phe                                                                               Leu                                                                               Arg                                                                               Asp                                                                               Glu                                                                               Tyr                                                                               Thr                                                                               Thr                            Leu                                                                               Lys                                                                               Glu                                                                               Thr                                                                               Trp                                                                               Asp                                                                               Arg                                                                               Ile                                                                               Leu                                                                               Ser                                                                               Thr                                                                               Asp                                                                               Val                                                                               Asp                                                                               Ala                                                                               Thr                                                                               Trp                            Gln                                                                               Trp                                                                               Lys                                                                               Asn                                                                               Phe                                                                               Ser                                                                               Gly                                                                               Leu                                                                               Gln                                                                               Glu                                                                               Val                                                                               Arg                                                                               Ser                                                                               His                                                                               Val                                                                               Pro                                                                               Lys                            Phe                                                                               Asp                                                                               Ala                                                                               Thr                                                                               Trp                                                                               Ala                                                                               Thr                                                                               Ala                                                                               Arg                                                                               Glu                                                                               Val                                                                               Thr                                                                               Leu                                                                               Lys                                                                               Thr                                                                               Phe                                                                               Ala                            Glu                                                                               Asp                                                                               Asn                                                                               Ser                                                                               Ala                                                                               Ser                                                                               Val                                                                               Gln                                                                               Ala                                                                               Thr                                                                               Met                                                                               Tyr                                                                               Lys                                                                               Met                                                                               Ala                                                                               Glu                                                                               Gln                            Ile                                                                               Leu                                                                               Ala                                                                               Arg                                                                               Gln                                                                               Gtn                                                                               Leu                                                                               Ile                                                                               Glu                                                                               Thr                                                                               Val                                                                               Glu                                                                               Tyr                                                                               Ser                                                                               Leu                                                                               Pro                                                                               Asn                            Lys                                                                               His                                                                               Tyr                                                                               Phe                                                                               Glu                                                                               Ile                                                                               Asp                                                                               Leu                                                                               Ser                                                                               Trp                                                                               His                                                                               Lys                                                                               Gly                                                                               Leu                                                                               Gln                                                                               Asn                                                                               Thr                            Gly                                                                               Lys                                                                               Asn                                                                               Ala                                                                               Glu                                                                               Val                                                                               Phe                                                                               Ala                                                                               Pro                                                                               Gln                                                                               Ser                                                                               Asp                                                                               Pro                                                                               Asn                                                                               Gly                                                                               Leu                                                                               Ile                            Lys                                                                               Cys                                                                               Thr                                                                               Val                                                                               Gly                                                                               Arg                                                                               Ser                                                                               Ser                                                                               Leu                                                                               Lys                                                                               Ser                                                                               Lys                                                                               Leu                                        __________________________________________________________________________

Because of the degeneracy of the genetic code, there are a large number of DNA sequences coding for a protein whose sequence corresponds to the formula given above. One preferred DNA sequence, particularly appropriate for an expression in the prokaryotic microorganisms, is as follows (SEQUENCE ID NO. 3):

    __________________________________________________________________________     ATGTCTGCGG                                                                              TAAAAGCAGC                                                                               GCGCTACGGC                                                                               AAGGACAATG                                                                               TTCGCGTCTA                              CAAGGTTCAC                                                                              AAGGACGAGA                                                                               AGACCGGTGT                                                                               CCAGACGGTG                                                                               TACGAGATGA                              CCGTCTGTGT                                                                              GCTTCTGGAG                                                                               GGTGAGATTG                                                                               AGACCTCTTA                                                                               CACCAAGGCC                              GACAACAGCG                                                                              TCATTGTCGC                                                                               AACCGACTCC                                                                               ATTAAGAACA                                                                               CCATTTACAT                              CACCGCCAAG                                                                              TAGAACCCCG                                                                               TTACTCCTCC                                                                               CGAGCTGTTC                                                                               GGCTCCATCC                              TGGGCACACA                                                                              CTTCATTGAG                                                                               AAGTACAACC                                                                               ACATCCATGC                                                                               CGCTCACGTC                              AACATTGTCT                                                                              GCCACCGCTG                                                                               GACCCGGATG                                                                               GACATTGACG                                                                               GCAAGCCACA                              CCCTCACTCC                                                                              TTCATCCGCG                                                                               ACAGCGAGGA                                                                               GAAGCGGAAT                                                                               GTGCAGGTGG                              ACGTGGTCGA                                                                              GGGCAAGGGC                                                                               ATCGATATCA                                                                               AGTCGTCTCT                                                                               GTCCGGCCTG                              ACCGTGCTGA                                                                              AGAGCACCAA                                                                               CTCGCAGTTC                                                                               TGGGGCTTCC                                                                               TGCGTGACGA                              GTACACCACA                                                                              CTTAAGGAGA                                                                               CCTGGGACCG                                                                               TATCCTGAGC                                                                               ACCGACGTCG                              ATGCCACTTG                                                                              GCAGTGGAAG                                                                               AATTTCAGTG                                                                               GACTCCAGGA                                                                               GGTCCGCTCG                              CACGTGCCTA                                                                              AGTTCGATGC                                                                               TACCTGGGCC                                                                               ACTGCTCGCG                                                                               AGGTCACTCT                              GAAGACTTTT                                                                              GCTGAAGATA                                                                               ACAGTGCCAG                                                                               CGTGCAGGCC                                                                               ACTATGTACA                              AGATGGCAGA                                                                              GCAAATCCTG                                                                               GCGCGCCAGC                                                                               AGCTGATCGA                                                                               GACTGTCGAG                              TACTCGTTGC                                                                              CTAACAAGCA                                                                               CTATTTCGAA                                                                               ATCGACCTGA                                                                               GCTGGCACAA                              GGGCCTCCAA                                                                              AACACCGGCA                                                                               AGAACGCCGA                                                                               GGTCTTCGCT                                                                               CCTCAGTCGG                              ACCCCAACGG                                                                              TCTGATCAAG                                                                               TGTACCGTCG                                                                               GCCGGTCCTC                                                                               TCTGAAGTCT                              AAATTG.                                                                        __________________________________________________________________________

Another preferred DNA sequence, which is particularly suitable for expression in eukaryotic cells, such as yeast, is as follows (SEQUENCE ID NO. 4):

    __________________________________________________________________________     ATGTCTGCTG                                                                              TTAAGGCTGC                                                                               TAGATACGGT                                                                               AAGGACAACG                                                                              TTAGAGTCTA                               CAAGGTTCAC                                                                              AAGGACGAGA                                                                               AGACCGGTGT                                                                               CCAGACGGTG                                                                              TACGAGATGA                               CCGTCTGTGT                                                                              GCTTCTGGAG                                                                               GGTGAGATTG                                                                               AGACCTCTTA                                                                              CACCAAGGCC                               GACAACAGCG                                                                              TCATTGTCGC                                                                               AACCGACTCC                                                                               ATTAAGAACA                                                                              CCATTTACAT                               CACCGCCAAG                                                                              CAGAACCCCG                                                                               TTACTCCTCC                                                                               CGAGCTGTTC                                                                              GGCTCCATCC                               TGGGCACACA                                                                              CTTCATTGAG                                                                               AAGTACAACC                                                                               ACATCCATGC                                                                              CGCTCACGTC                               AACATTGTCT                                                                              GCCACCGCTG                                                                               GACCCGGATG                                                                               GACATTGACG                                                                              GCAAGCCACA                               CCCTCACTCC                                                                              TTCATCCGCG                                                                               ACAGCGAGGA                                                                               GAAGCGGAAT                                                                              GTGCAGGTGG                               ACGTGGTCGA                                                                              GGGCAAGGGC                                                                               ATCGATATCA                                                                               AGTCGTCTCT                                                                              GTCCGGCCTG                               ACCGTGCTGA                                                                              AGAGCACCAA                                                                               CTCGCAGTTC                                                                               TGGGGCTTCC                                                                              TGCGTGACGA                               GTACACCACA                                                                              CTTAAGGAGA                                                                               CCTGGGACCG                                                                               TATCCTGAGC                                                                              ACCGACGTCG                               ATGCCACTTG                                                                              GCAGTGGAAG                                                                               AATTTCAGTG                                                                               GACTCCAGGA                                                                              GGTCCGCTCG                               CACGTGCCTA                                                                              AGTTCGATGC                                                                               TACCTGGGCC                                                                               ACTGCTCGCG                                                                              AGGTCACTCT                               GAAGACTTTT                                                                              GCTGAAGATA                                                                               ACAGTGCCAG                                                                               CGTGCAGGCC                                                                              ACTATGTACA                               AGATGGCAGA                                                                              GCAAATCCTG                                                                               GCGCGCCAGC                                                                               AGCTGATCGA                                                                              GACTGTCGAG                               TACTCGTTGC                                                                              CTAACAAGCA                                                                               CTATTTCGAA                                                                               ATCGACCTGA                                                                              GCTGGCACAA                               GGGCCTCCAA                                                                              AACACCGGCA                                                                               AGAACGCCGA                                                                               GGTCTTCGCT                                                                              CCTCAGTCGG                               ACCCCAACGG                                                                              TCTGATCAAG                                                                               TGTACCGTCG                                                                               GCCGGTCCTC                                                                              TCTGAAGTCT                               AAATTG.                                                                        __________________________________________________________________________

Another preferred DNA sequence, which is notably suitable for expression in animal cells, is as follows (SEQUENCE ID NO. 6):

    __________________________________________________________________________                 5'-ATGTC                                                                             CGCAGTAAAA                                                                              GCAGCCCGCT                                                                               ACGGCAAGGA                                CAATGTCCGC                                                                              GTCTACAAGG                                                                              TTCACAAGGA                                                                              CGAGAAGACC                                                                               GGTGTCCAGA                                CGGTGTACGA                                                                              GATGACCGTC                                                                              TGTGTGCTTC                                                                              TGGAGGGTGA                                                                               GATTGAGACC                                TCTTACACCA                                                                              AGGCCGACAA                                                                              CAGCGTCATT                                                                              GTCGCAACCG                                                                               ACTCCATTAA                                GAACACCATT                                                                              TACATCACCG                                                                              CCAAGCAGAA                                                                              CCCCGTTACT                                                                               CCTCCCGAGC                                TGTTCGGCTC                                                                              CATCCTGGGC                                                                              ACACACTTCA                                                                              TTGAGAAGTA                                                                               CAACCACATC                                CATGCCGCTC                                                                              ACGTCAACAT                                                                              TGTCTGCCAC                                                                              CGCTGGACCC                                                                               GGATGGACAT                                TGACGGCAAG                                                                              CCACACCCTC                                                                              ACTCCTTCAT                                                                              CCGCGACAGC                                                                               GAGGAGAAGC                                GGAATGTGCA                                                                              GGTGGACGTG                                                                              GTCGAGGGCA                                                                              AGGGCATCGA                                                                               TATCAAGTCG                                TCTCTGTCCG                                                                              GCCTGACCGT                                                                              GCTGAAGAGC                                                                              ACCAACTCGC                                                                               AGTTCTGGGG                                CTTCCTGCGT                                                                              GACGAGTACA                                                                              CCACACTTAA                                                                              GGAGACCTGG                                                                               GACCGTATCC                                TGAGCACCGA                                                                              CGTCGATGCC                                                                              ACTTGGCAGT                                                                              GGAAGAATTT                                                                               CAGTGGACTC                                CAGGAGGTCC                                                                              GCTCGCACGT                                                                              GCCTAAGTTC                                                                              GATGCTACCT                                                                               GGGCCACTGC                                TCGCGAGGTC                                                                              ACTCTGAAGA                                                                              CTTTTGCTGA                                                                              AGATAACAGT                                                                               GCCAGCGTGC                                AGGCCACTAT                                                                              GTACAAGATG                                                                              GCAGAGCAAA                                                                              TCCTGGCGCG                                                                               CCAGCAGCTG                                ATCGAGACTG                                                                              TCGAGTACTC                                                                              GTTGCCTAAC                                                                              AAGCACTATT                                                                               TCGAAATCGA                                CCTGAGCTGG                                                                              CACAAGGGCC                                                                              TCCAAAACAC                                                                              CGGCAAGAAC                                                                               GCCGAGGTCT                                TCGCTCCTCA                                                                              GTCGGACCCC                                                                              AACGGTCTGA                                                                              TCAAGTGTAC                                                                               CGTCGGCCGG                                TCCTCTCTGA                                                                              AGTCTAAATT                                                                              G                                                            __________________________________________________________________________

preceded by a non-translated 5' sequence favoring expression in animal cells. A preferred non-translated 5' sequence of this type is the one comprising the sequence (SEQUENCE ID NO. 5) AGCTTGCCGCCACT, located immediately upstream from the sequence described above.

It will be noticed that the protein coded for by the cDNA sequences given above can undergo processing by methionyl aminopeptidase, which cleaves it from its amino-terminal methionine residue.

The invention further relates to an expression vector carrying the above-defined recombinant gene with the means necessary for its expression.

For expression in prokaryotic microorganisms, in particular in Escherichia coli, the coding sequence must be inserted into an expression vector containing especially an effective promoter, followed by a ribosome binding site upstream from the gene to be expressed, and also an effective transcription stop sequence downstream from the gene to be expressed. This plasmid must also contain an origin of replication and a selection marker. All these sequences must be chosen as a function of the host cell.

For expression in eukaryotic cells, the expression vector according to the invention carries the above-defined recombinant gene with the means necessary for its expression, for its replication in eukaryotic cells and for selection of the transformed cells. Preferably, this vector carries a selection marker, chosen for example to complement a mutation of the recipient eukaryotic cells, which makes it possible to select those cells which have integrated a large number of copies of the recombinant gene either into their genome or into a multicopy vector.

For expression in animal cells, especially in the cells of Chinese hamster ovaries, CHO, the coding sequence is inserted into a plasmid (for example derived from pBR322) containing two expression units, a first unit, into which the recombinant gene is inserted, before an effective promoter (for example the SV40 early promoter). The sequence around the initiation ATG is preferably chosen as a function of the consensus sequence described by KOZAK (M. KOZAK (1978) Cell, 15, 1109-1123). An intron sequence, for example the intron of mouse α-globin, can be inserted upstream from the recombinant gene, and a sequence containing a polyadenylation site, for example an SV40 polyadenylation sequence, can be inserted downstream from the recombinant gene. The second expression unit contains a selection marker (for example a DNA sequence) coding for dihydrofolate reductase (an enzyme abbreviated hereafter to DHFR). The plasmid is transfected in animal cells, for example DHFR- CHO cells (incapable of expressing DHFR). A line is selected for its methotrexate resistance: it has integrated a large number of copies of the recombinant into its genome and expresses said recombinant gene at a sufficient level.

For expression in eukaryotic cells such as yeast, for example Saccharomyces cerevisiae, the coding sequence should be inserted between, on the one hand, sequences recognized as an effective promoter and, on the other hand, a transcription terminator. The array promoter/coding sequence/terminator, which is called an expression cassette, is either cloned in a plasmid vector (single-copy or multicopy) for the yeast, or integrated as a multicopy into the genome of the yeast.

The invention further relates to the eukaryotic cells transformed by the above expression vector. Of value among these eukaryotic cells are strains of the species Saccharomyces cerevisiae, in particular those which contain a mutation on one of the genes responsible for the synthesis of leucine or uracil, for example the LEU2 gene or the URA3 gene.

The invention further relates to the animal cells containing this recombinant gene with the means necessary for its expression. Said recombinant gene may, for example, have been introduced into the cells by transfection by the above expression vector, by infection with a virus or a retrovirus carrying said expression vector, or by microinjection.

The invention further relates to the process for producing a recombinant urate oxidase which comprises the steps of:

1) cultivating transformed cells as hereinabove defined;

2) producing the lysis of that cells;

3) isolating and purifying the urate oxidase contained in the obtained lysate.

The invention will be understood more clearly with the aid of the Examples below.

Many of the following techniques, which are well known to those skilled in the art, are described in detail in the work by Maniatis et al.: "Molecular cloning: a laboratory manual" published in 1984 by Cold Spring Harbor Press in New York.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an elution profile by measurement of the optical density at 218 nm of the product of tryptic digestion of urate oxidase.

FIG. 2 shows an elution profile by measurement of the optical density at 218 nm of the product of digestion of urate oxidase with protease V8.

FIG. 3 shows a nucleotide sequence of clone 9C and of part of clone 9A.

FIG. 4 shows a DNA sequence opened by ATG in position 109 in FIG. 3 and polypeptide coded for. The sequenced peptides obtained by lysis of A. Flavus urate oxidase with trypsin (⃡) and protease V8 ( ) are shown by arrows opposite the polypeptide coded for.

FIG. 5 shows plasmid p163,1.

FIG. 6 shows plasmid p160.

FIG. 7 shows plasmid p373,2.

FIG. 8 shows plasmid p462.

FIG. 9 shows plasmid p466.

FIG. 10 shows plasmid pEMR414.

FIG. 11 shows plasmid pEMR469.

FIG. 12 shows plasmid pEMR473.

FIG. 13 shows plasmid PSE₁.

FIG. 14 shows plasmid pSV860.

EXAMPLE 1: Isolation of the messenger RNA's from Aspergillus flavus

The strain of A. flavus which produces urate oxidase was cultivated under conditions appropriate for the production of urate oxidase, i.e. in a medium containing uric acid and having the following composition: glucose 15 g/l, MgSO₄.7H₂ O 1 g/l, KH₂ PO₄ 0.75 g/l, CaCO₃ 1.2 g/l, uric acid 1.2 g/l, KOH 0.5 g/l, soy bean oil 0.66 ml/l, FeSO₄.7H₂ O 10 mg/l, CuSO₄.5H₂ O 1 mg/l, ZnSO₄.7H₂ O 3 mg/l, MnSO₄.H₂ O 1 mg/l. The medium is adjusted to pH 7 with H₂ SO₄ 1M and sterilized at 120° C. for 80 min.

In a 5 l Erlenmeyer flask, 1.5 l of medium are inoculated with about 1 to 3.10⁷ spores.

The culture is incubated for about 40 h at 30° C., with agitation (120 rpm). The mycelium is recovered by filtration on gauze, washed with water and frozen in liquid nitrogen.

15 g of mycelium (wet weight) are thawed, resuspended in 45 ml of lysis buffer and then taken up in the same volume of beads (0.45 μm in diameter). The lysis buffer consists of guanidine thiocyanate 4M, Tris-HCl 10 mM pH 7.6, EDTA 10 mM, β-mercaptoethanol 50 ml/l. The mycelian suspension is ground in a Zellmuhler mill (vibrogenic) for 5 min.

The ground material is recovered and the beads are decanted. The supernatant is removed (about 45 ml), brought back to a final concentration of 3M in respect of lithium chloride and stored at 0° C.

After two days, it is centrifuged for 60 min at 10,000 rpm. The supernatant is discarded and the residue is taken up in 40 ml of LiCl 3M and centrifuged again at 10,000 rpm for 1 h 30 min.

The following are added: proteinase K (SIGMA) 40 μg/ml, SDS (0.1% w/v) and EDTA 20 mM. The mixture is incubated at 37° C. for 3 h. Precipitation with 2 volumes of ethanol is followed by washing with 70% ethanol. The residue is taken up in 0.5 ml of TE buffer (Tris-HCl 10 mM, EDTA 1 mM pH 7.5), the mixture is extracted twice with chloroform and precipitation is carried out with ethanol. The RNA's are stored at -80° C. in alcohol.

EXAMPLE 2: Purification of the poly A⁺ fraction of the RNA's

About 1 mg of RNA is precipitated for 20 min at 4° C. (15,000 rpm) and then washed with 70% ethanol and dried. The residue is taken up in 1 ml of TE buffer and resuspended by agitation in a Vortex. Oligo dT-cellulose type 3 (marketed by Collaborative Research Inc., Biomedicals Product Division) is prepared according to the manufacturer's recommendations. The RNA is deposited on the oligo dT, agitated gently to resuspend the beads and then heated for 1 min at 65° C.

The suspension is adjusted to 0.5M NaCl and then agitated gently for 10 min. It is then centrifuged for 1 min at 1000 rpm, the supernatant is removed and the residue is washed twice with 1 ml of TE buffer containing 0.5M NaCl. The supernatants are removed. The polyadenylated fraction of the RNA's (consisting of the messenger RNA's) is eluted by suspending the beads in 1 ml of TE buffer, then heating this suspension at 60° C. for 1 min and subsequently agitating it for 10 min on a tilting plate. It is then centrifuged for 1 min at 1000 rpm, which makes it possible to recover on the one hand the supernatant containing free mRNA's in solution, and on the other hand the residue of cellulose beads. The above series of operations (starting from elution) is repeated. The supernatants obtained in this way are pooled, the excess beads are removed by centrifugation and the supernatant is precipitated with ethanol containing NaCl in accordance with the usual techniques (Maniatis: op. cit.).

EXAMPLE 3: Building of the cDNA library

The messenger RNA's isolated as described in the previous Example were used to build a cDNA library in vector pTZ19R (marketed by PHARMACIA). This vector is a plasmid comprising a polylinker containing unique restriction sites.

The cloning technique used is the one described by Caput et al. (primer-adapter technique: Caput et al., Proc. Natl. Acad. Sci. (U.S.A.) (1986) 83, 1670-1674).

It consists firstly in digesting the vector with Pst1, adding a polydC tail to the protuberant 3' end and then digesting the resulting plasmids with BamHI. The fragment corresponding to the vector is purified on a column of Sepharose CL4B (Pharmacia). It therefore comprises a polydC tail at one end, the other end being a sticky end of the BamHI type. Secondly, the messenger RNA's are subjected to reverse transcription starting from a primer having the sequence (SEQUENCE ID NO. 7) 5' <GATCCGGGCCCT.sub.(12))<3. Thus the cDNA's have at their 5' end the sequence GATCC complementary to the BamHI sticky end. The RNA-DNA hybrids obtained by the action of reverse transcriptase are subjected to alkaline hydrolysis, enabling the RNA to be removed. The single-stranded cDNA's are then purified by 2 cycles on a column of Sepharose CL4B and subjected to a treatment with terminal transferase so as to add polydG's at the 3' end. The cDNA's are inserted in single-stranded form into the vector prepared as described above. A second oligonucleotide, the adapter, complementary to the primer, is necessary in order to generate an "open" BamHI site at the 5' end of the cDNA's. After hybridization of the vector, the cDNA and the adapter, the recombinant molecules are circularized by the action of the ligase of phage T4. The single-stranded regions are then repaired by means of the DNA polymerase of phage T4. The plasmid pool obtained in this way is used to transform the MC1061 strain for ampicillin resistance (Casabadan, Chou and Cohen, J. Bact. (1980) 143, pages 971-980.

EXAMPLE 4: Purification of urate oxidase extracted from A. flavus and characterization thereof 1) Purification of urate oxidase extracted from A. flavus

A preparation of urate oxidase extracted from A. flavus (Uricozyme-Laboratoires Clin Midy), having a specific urate oxidase activity of 8 U/ml (the specific urate oxidase activity is the ratio of the urate oxidase activity measured by the test described in Example 9 to the weight of total proteins measured by the Bradford method: Anal. Biochem., 72, 248-254), was repurified by chromatography on a column of Red-agarose 120 grafted agarose (SIGMA), concentration by ultrafiltration and filtration on Ultrogel Aca 44 (IBF), a polyacrylamideagarose gel, according to the following protocol:

Step 1: Affinity chromatography on grafted agarose

Temperature: 4° C.

Column: PHARMACIA K50/30

diameter=50 mm

length=33 cm

Resin: Red 120 Agarose (3.000 CL/R-0503 SIGMA)

(volume of gel=410 ml

height of gel=20 cm)

Equilibration buffer: glycine/NaOH 20 mM pH 8.3

Elution buffer: glycine/NaOH 20 mM, NaCl 2M pH 8.3

Conditioning flow rate: 250 ml.h⁻¹

Operating flow rate: 160 ml.h⁻¹

Elution flow rate: 60 ml.h⁻¹

1) Deposit the solution of Uricozyme on the top of the column with the aid of a constant-flow pump.

2) After adsorption, wash the column with twice its volume of equilibration buffer.

3) Elute with an ionic strength gradient having the following composition:

glycine, NaOH, 20 mM pH 8.3/glycine, NaOH, 20 mM+NaCl 2M pH8.3

The total volume of the gradient is equal to 10 times the volume of the column, divided up equally between the two constituents.

Chromatographic recording is carried out at λ=280 nm; the urate oxidase pool is collected after combination of the fractions which have a specific urate oxidase activity greater than or equal to 16 U/mg.

Step 2: Concentration of the urate oxidase pool by ultrafiltration with the aid of a Biopass system comprising a 10 kDa ultrafiltration membrane

Step 3:

Temperature: 4° C.

Column: PHARMACIA K 50/100

diameter=50 mm

length=100 cm

Resin: polyacrylamide-agarose with amine and hydroxyl groups: Ultrogel ACA 44 (IBF)

volume of gel=1.6 l

height of gel=80 cm

Equilibration buffer: glycine/NaOH 20 mM pH 8.3

Conditioning flow rate: 40 ml.h⁻¹

Operating flow rate: 24 ml.h³¹ 1

1) Deposit the concentrated urate oxidase pool on the top of the column with the aid of a constant-flow pump.

2) After the sample has been deposited, continue to supply the column with the buffer glycine/NaOH 20 mM pH 8.3

3) After chromatography, wash with NaCl 2M until the UV absorbance value (λ=280 nm)<0.05.

Store under NaCl 2M at 4° C.

Chromatographic recording is carried out at λ=280 nm; the urate oxidase pool is collected after combination of the fractions which cojointly have:

a specific urate oxidase activity greater than or equal to 20 U/mg; and

only 2 bands in electrophoresis under denaturing conditions (presence of SDS) and with silver nitrate developing (Biorad staining kit), namely:

a major band of 33-34 kDa

a minor band of 70-71 kDa.

2) Characterization of purified urate oxidase extracted from A. flavus

a) Partial sequencing

Direct amino-terminal sequencing of the protein was attempted in order to obtain information on the amino acid sequence of the purified urate oxidase extract, making it possible to synthesize the probes necessary for cloning the cDNA. This sequencing was not successful because of amino-terminal blocking of the protein (cf. f) below).

The following strategy was therefore developed to obtain the partial sequence of urate oxidase:

cleavage of the protein with proteolytic enzymes (using the enzymes trypsin and protease V8 of Staphylococcus aureus

separation of the resulting polypeptides by reversed phase HPLC

sequencing of the purified peptides.

α) Hydrolysis of the urate oxidase with trypsin, purification and sequencing of the peptides

The urate oxidase, at a concentration of 9 mg/ml in an ammonium carbonate buffer 100 mM pH 8.9, was digested with trypsin (Worthington, TPCK), in a ratio urate oxidase/trypsin of 30/1 by weight, at 30° C. for 24 h. After tryptic hydrolysis, 60 μg of digested urate oxidase were directly injected on to a reversed phase HPLC column of Brownlee G18 grafted silica (column: 10×0.2 cm) equilibrated with acetonitrile 1% (v/v) and trifluoroacetic acid 0.1% (v/v) in water. The peptides were then eluted by a linear gradient of acetonitrile in a solution of trifluoroacetic acid (0.1% v/v) in water, varying from 1% to 60% of acetonitrile in 60 min, at a rate of 150 μl/min. The peptides leaving the column were detected by measurement of the optical density at 218 nm.

The elution profile is shown in FIG. 1, in which the numbers following the letter T (trypsin) correspond to the peaks identified.

Each peak was collected and stored at -20° C. until analyzed on a protein sequencer (model 470 A from Applied Biosystems) equipped with a chromatograph (model 430 A from Applied Biosystems), which continuously analyzes the phenylthiohydantoic derivatives formed, after each degradation cycle. Table I below shows the peptide sequences of the 9 peaks identified which have been assigned SEQUENCE ID NOs. as follows: T 17-SEQUENCE ID NO. 8; T 20-SEQUENCE ID NO. 9; T 23-SEQUENCE ID NO. 10; T 27-SEQUENCE ID NO. 11; T 28-SEQUENCE ID NO. 12; T 29-SEQUENCE ID NO. 13; T 31-SEQUENCE ID NO. 14; T 32-SEQUENCE ID NO. 15; T 33-SEQUENCE ID NO. 16.

β) Hydrolysis of the urate oxidase with protease V8, purification and sequencing of the peptides

The urate oxidase, at a concentration of 2 mg/ml in an ammonium acetate buffer 100 mM pH 6.8, was digested with the protease V8 of Staphylococcus aureus (Boehringer-Mannheim), in a ratio urate oxidase/protease V8 of 60/1, at 30° C. for 72 h. 160 μg of digested urate oxidase were then injected on to a reversed phase HPLC column of Brownlee G18 grafted silica (column: 10×0.2 cm; particles: 7×0.03 μm), equilibrated with acetonitrile 1% and trifluoroacetic acid 0.1% (v/v) in water. The peptides were then eluted by a linear gradient of acetonitrile in a solution of trifluoroacetic acid in water (0.1% (v/v)), varying from 1% to 60% of acetonitrile in 60 min, at a rate of 150 μl/min. The peptides leaving the column were detected by measurement of the optical density at 218 nm.

The elution profile is shown in FIG. 2, in which the numbers following the letter V (protease V8) correspond to the peaks identified.

Each peak was collected and stored at -20° C. until analyzed on the protein sequencer already mentioned.

Table I below shows the peptide sequences of the 5 peaks identified which have been assigned SEQUENCE ID NOs. as follows: V 1-SEQUENCE ID NO. 17; V 2-SEQUENCE ID NO. 18; V 3-SEQUENCE ID NO. 19; V 5-SEQUENCE ID NO. 20; and V 6-SEQUENCE ID NO. 21.

                                      TABLE I                                      __________________________________________________________________________               Sequencing of the products obtained by hydrolysis                    __________________________________________________________________________     With the aid                                                                          T 17                                                                              Asn - Val - Gln - Val - Asp - Val - Val - Glu - Gly - Lys            of trypsin                                                                            T 20                                                                              Asn - Phe - Ser - Gly - Leu - Gln - Glu - Val                               T 23                                                                              Phe - Asp - Ala - Thr - Trp - Ala                                           T 27                                                                              His - Tyr - Phe - Glu - Ile - Asp - Leu - Ser                               T 28                                                                              Ile - Leu - Ser - Thr - Asp - Val - Asp - Ala - Thr - Trp - Gln                - Trp - Lys                                                                 T 29                                                                              His - Tyr - Phe - Glu - Ile - Asp - Leu - Ser - Trp - His -                    Lys                                                                         T 31                                                                              Ser - Thr - Asn - Ser - Gln - Phe - Trp - Gly - Phe - Leu -                    Arg                                                                         T 32                                                                              Gln - Asn - Pro - Val - Thr - Pro - Pro - Glu - Leu - Phe - Gly                - Ser - Ile -                                                                  Leu - Gly - Thr                                                             T 33                                                                              Gln - Asn - Pro - Val - Thr - Pro - Pro - Glu - Leu - Phe - Gly                - Ser - Ile -                                                                  Leu - Gly - Thr                                                      With the aid                                                                          V 1                                                                               Tyr - Ser - Leu - Pro - Asn - Lys - His - Tyr - Phe - Glu - Ile                - Asp - Leu -                                                        of protease                                                                              Ser - Trp - His - Lys                                                V8     V 2                                                                               Val - Thr - Leu - Lys - Thr - Phe - Ala - Glu - Asp - Asn - Ser                - Ala - Ser -                                                                  Val - Gln - Ala                                                             V 3                                                                               Thr - Ser - Tyr - Thr - Lys - Ala - Asp - Asn - Ser - Val - Ile                - Val - Asp -                                                                  Thr - Asp - Ser - Ile - Lys - Asn - Thr - Ile - Tyr - Ile -                    Thr                                                                         V 5                                                                               Gly - Lys - Gly - Ile - Asp - Ile - Lys - Ser - Ser - Leu - Ser                - Gly - Leu -                                                                  Thr - Val - Leu - Lys - Ser - Thr - Asn - Ser - Gln - Phe - Trp                - Gly - Phe -                                                                  Leu - Arg                                                                   V 6                                                                               Gly - Lys - Gly - Ile - Asp - Ile - Lys - Ser - Ser - Leu - Ser                - Gly - Leu -                                                                  Thr - Val - Leu - Lys                                                __________________________________________________________________________

b) Specific activity

The purified urate oxidase extract has a specific activity of about 30 U/mg.

c) Electrophoresis under denaturing conditions

Electrophoresis of the purified urate oxidase extract on polyacrylamide gel in the presence of SDS (sodium dodecylsulfate), followed by silver developing, reveals a high intensity band of about 33-34 kDa and a very low intensity band of about 70-71 kDa.

d) Determination of the isoelectric point

Procedure

Use of ready-to-use gels, namely LKB Ampholines gel plates from Pharmacia with pH ranges of (3.5-9.5) and (5-8).

Deposition of 10 μl of LKB standard proteins (range of isoelectric points of the standard proteins: 3.5-9.5) and 4 μg and 8 μg of purified urate oxidase (on two different lanes).

Run 1 h 30 min, 12 V, 6° C.

Then staining with Coomassie blue (0.1%) in (25% ethanol, 8% acetic acid) to stain the proteins, followed by decolorization with a solution containing 25% of ethanol and 8% of acetic acid (to eliminate the background).

Results: Observation of two close bands (doublet), of isoelectric points 8.1 and 7.9, on each of the two lanes.

e) Two-dimensional gel analysis

Two-dimensional gel analysis makes it possible to separate the proteins in a first stage according to their isoelectric points and in a second stage according to their molecular weights.

Protocol

Sample: solution of purified urate oxidase extract in a glycine buffer 20 mM pH 8.3

Preparation of the sample

Two samples of 5 μg and 10 μg of urate oxidase.

Drying by vacuum centrifugation and taking-up in 5 μl of a lysis buffer having the following composition: urea 2.5M, 3-(3-cholamidopropyl)dimethylammoniopropane-1-sulfonate, CHAPS (Sigma), 2% (v/v), Ampholines amphoterics (LKB) of pH ranges 5-8 and 3.5-9.5, 0.4%, and β-mercaptoethanol 5%.

Isoelectrofocusing gel

Preparation of a solution containing urea 9.5M, CHAPS 5%, LKB Ampholines (pH (3.5-9.5) 1%; pH (5-8) 1%), acrylamide/bisacrylamide (28.4%/1.7%) 3.5% final concentration, H₂ O.

Filtration and degassing of the solution, followed by addition of 0.075% of tetramethylethylenediamine, Temed (Pharmacia), and 0.015% of ammonium persulfate.

Introduction of the solution into tubes (16×0.12 cm)-polymerization overnight at 20° C.

Cathodic solution: NaOH 0.1M, degassed.

Anodic solution: H₃ PO₄ 25 mM.

Prerun 45 min at 4 mA (voltage 300 V→1000 V).

Deposition of the samples at the cathode.

Run 19 h at 1000 V and at 20° C.

Demolding of the gels and equilibration for 10 min at 20° C. in a buffer (Tris 0.375M pH 8.8; SDS 3%; dithiothreitol, DTT, 50 mM).

PAGE/SDS denaturing gel

Preparation of a solution containing acrylamide/bisacrylamide (30%/0.8%) 15% final concentration, Tris-HCl (pH 8.8) 0.375M, H₂ O.

Filtration and degassing of the solution, followed by addition of SDS (0.1%), ammonium persulfate 0.05% and Temed 0.05%.

Polymerization overnight at 4° C. (gel 16×20×0.15 cm).

After equilibration, deposition of the isoelectro-focusing gel on the surface of the PAGE/SDS gel, followed by sealing with agarose.

Electrophoresis buffer: (Tris-HCl 25 mM pH 8.3, glycine 0.192M, SDS 0.1%).

Run 100 mA-6 h at 6° C.

Fixing of the gel in 50% methanol, 10% acetic acid, followed by silver nitrate staining (method of Blum, H., Electrophoresis 1987, 8, p. 93-99).

Scanning of the gel on a Visage 2000 image analyzer from Kodak for determination of the optical density and surface area of each spot and hence for calculation of the quantitative ratio between the spots.

Determination of the molecular weight of the protein by preparation of a two-dimensional gel in the presence of Amersham standard proteins.

Result

Two spots with a molecular weight of the order of 33.5 kDa are observed, one being the majority spot with an isoelectric point of the order of 8.0, intensity 5.2 (representing about 93% of the weight of proteins), and the other being the minority spot with an isoelectric point of the order of 7.4, intensity 0.41 (representing about 7% of the weight of proteins).

f) Determination of the amino-terminal sequence and the mass of the blocking amino-terminal group

α) Demonstration of the blocked character of the amino-terminal sequence

The amino-terminal sequence was analyzed with the aid of an Applied Biosystem model 470A sequencer coupled with an Applied Biosystem model 120A analyzer of phenylthiohydantoic derivatives. The purified urate oxidase (200 pmol, checked by amino acid analysis) was deposited on the sequencer in the presence of 20 pmol of β-lactoglobulin, a standard protein.

No amino-terminal sequence corresponding to a urate oxidase sequence was detected (by contrast, the amino-terminal sequence of the standard protein was detected, showing that the sequencer was working).

A. flavus urate oxidase therefore has the amino-terminal end blocked.

β) Determination of the sequence of an amino-terminal peptide of 32 amino acids and the mass of the blocking amino-terminal group Method: Digestion with cyanogen bromide

The purified urate oxidase extract is subjected to gel filtration on Sephadex G25 (PD10-Pharmacia), a gel obtained by crosslinking dextran with epichlorohydrin, equilibrated with a solution containing 7% of formic acid, making it possible to remove the salts and change the buffer. The formic acid concentration is increased to 70% by vacuum centrifugation. Cyanogen bromide is then added to a final concentration of 0.2M and the reaction is allowed to proceed for 20 h under argon, in the absence of light and at room temperature.

Separation by ion exchange chromatography of the peptides derived from digestion of the protein with cyanogen bromide

The peptides were separated on an ion exchange column based on mono S hydrophilic resin (Pharmacia).

Buffer A: ammonium acetate 10 mM pH 6.2

Buffer B: ammonium acetate 1M pH 6.2

Flow rate: 0.6 ml/min, peak detection by measurement of the optical density at 278 nm

Gradient: from 0% of B to 100% of B in 30 min-collection of 1 ml fractions

The fractions derived from the ion exchange step were analyzed by PAGE/SDS gel according to the method described by Schagger and Von Jagow (1987) Anal. Biochem. 166-p. 368-379.

Purification of the amino-terminal peptide by reversed phase HPLC and analysis thereof by mass spectrometry

The peptide derived from the ion exchange step, having a molecular weight of about 4000 Da (on PAGE/SDS gel), was purified on a Beckman Altex C18 column (250×2.1 mm), which is a reversed phase HPLC column based on C18 grafted silica.

Flow rate: 0.3 ml/min, peak detection by measurement of the optical density at 218 nm

Buffer A: H₂ O/0.1% TFA (trifluoroacetic acid)

Buffer B: acetonitrile/0.1% TFA

Gradient: from 1 to 50% of B in 60 min.

The peptide collected after a first reversed phase HPLC step was repurified on the same reversed phase HPLC column, but with a different gradient.

Gradient: from 1 to 50% of B in 10 min.

The peak collected was subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS) with a glycerol+thioglycerol matrix.

Digestion of the amino-terminal peptide with chymotrypsin and amino acid analysis of the chymotryptic peptides separated by reversed phase HPLC

To establish the sequence of the peptide purified by reversed phase HPLC, said peptide was digested with chymotrypsin. The chymotryptic peptides were separated by reversed phase HPLC on a Beckman Altex C18 column (250×2.1 mm).

Flow rate: 0.3 ml/min, peak detection by measurement of the optical density at 218 nm

Buffer A: H₂ O/0.11% TFA

Buffer B: acetonitrile/0.08% TFA

Gradient: from 1% of B to 50% of B in 60 min-collection of the peeks.

The chymotryptic peptides were identified by amino acid analysis on an Applied Biosystem analyzer (model 420-130A).

Results

The results presented below, which were established after determination of the sequence of the cDNA of A. flavus urate oxidase and the deduced amino acid sequence (cf. Example 6), can only be understood in the light of the following:

Analysis of the amino-terminal peptide by mass spectrometry

A difference of about 42 atomic mass units is observed between the two molecular weights determined by mass spectrometry, 3684 and 3666, and the theoretical molecular weights determined from the following sequence (amino acid sequence deduced from the cDNA of A. flavus urate oxidase with cleavage of the amino-terminal methionine group and peptide cleavage with cyanogen bromide after the first methionine residue) which corresponds to amino acids 1-31 of SEQUENCE ID NO. 1: ##STR1## with a carboxy-terminal methionine residue modified by reaction with cyanogen bromide to give either homoserine, 3642, or homoserine lactone, 3624.

There is therefore a blocking group on the amino-terminal serine which accounts for an additional mass of about 42 atomic mass units, probably corresponding to acetylation of said amino-terminal serine (mass of CH₃ CO-mass of H=42 atomic mass units).

Amino acid analysis of the chymotryptic peptides

This analysis made it possible to show unambiguously that the sequence of the amino-terminal peptide obtained by digestion with cyanogen bromide comprises the sequence (1) described above.

The complete amino acid sequence of urate oxidase is shown hereinafter (SEQUENCE ID NO. 1).

    ______________________________________                                         Ser  Ala    Val    Lys  Ala  Ala  Arg  Tyr  Gly  Lys                           Asp  Asn    Val    Arg  Val  Tyr  Lys  Val  His  Lys                           Asp  Glu    Lys    Thr  Gly  Val  Gln  Thr  Val  Tyr                           Glu  Met    Thr    Val  Cys  Val  Leu  Leu  Glu  Gly                           Glu  Ile    Glu    Thr  Ser  Tyr  Thr  Lys  Ala  Asp                           Asn  Ser    Val    Ile  Val  Ala  Thr  Asp  Ser  Ile                           Lys  Asn    Thr    Ile  Tyr  Ile  Thr  Ala  Lys  Gln                           Asn  Pro    Val    Thr  Pro  Pro  Glu  Leu  Phe  Gly                           Ser  Ile    Leu    Gly  Thr  His  Phe  Ile  Glu  Lys                           Tyr  Asn    His    Ile  His  Ala  Ala  His  Val  Asn                           Ile  Val    Cys    His  Arg  Trp  Thr  Arg  Met  Asp                           Ile  Asp    Gly    Lys  Pro  His  Pro  His  Ser  Phe                           Ile  Arg    Asp    Ser  Glu  Glu  Lys  Arg  Asn  Val                           Gln  Val    Asp    Val  Val  Glu  Gly  Lys  Gly  Ile                           Asp  Ile    Lys    Ser  Ser  Leu  Ser  Gly  Leu  Thr                           Val  Leu    Lys    Ser  Thr  Asn  Ser  Gln  Phe  Trp                           Gly  Phe    Leu    Arg  Asp  Glu  Tyr  Thr  Thr  Leu                           Lys  Glu    Thr    Trp  Asp  Arg  Ile  Leu  Ser  Thr                           Asp  Val    Asp    Ala  Thr  Trp  Gln  Trp  Lys  Asn                           Phe  Ser    Gly    Leu  Gln  Glu  Val  Arg  Ser  His                           Val  Pro    Lys    Phe  Asp  Ala  Thr  Trp  Ala  Thr                           Ala  Arg    Glu    Val  Thr  Leu  Lys  Thr  Phe  Ala                           Glu  Asp    Asn    Ser  Ala  Ser  Val  Gln  Ala  Thr                           Met  Tyr    Lys    Met  Ala  Glu  Gln  Ile  Leu  Ala                           Arg  Gln    Gln    Leu  Ile  Glu  Thr  Val  Glu  Tyr                           Ser  Leu    Pro    Asn  Lys  His  Tyr  Phe  Glu  Ile                           Asp  Leu    Ser    Trp  His  Lys  Gly  Leu  Gln  Asn                           Thr  Gly    Lys    Asn  Ala  Glu  Val  Phe  Ala  Pro                           Gln  Ser    Asp    Pro  Asn  Gly  Leu  Ile  Lys  Cys                           Thr  Val    Gly    Arg  Ser  Ser  Leu  Lys  Ser  Lys                           Leu                                                                            ______________________________________                                    

EXAMPLE 5: Screening of the bacteria 1) Preparation of the labeled probes

Two pools of probes deduced from amino acid sequences of the protein were synthesized with the aid of a Biosearch 4600 DNA synthesizer. The first pool corresponds to the sequence of residues which correspond to amino acids 1-6 of SEQUENCE ID NO. 11 His-Tyr-Phe-Glu-Ile-Asp (part of the sequence of T 27), i.e. from 5' to 3': ##STR2## This pool in fact consists of 2⁴ ×3=48 different oligonucleotides, representing all the possible combinations. The second pool corresponds to the sequence of amino acid residues which correspond to amino acids 22-27 of SEQUENCE ID NO. 20 Gln-Phe-Trp-Gly-Phe-Leu (part of the sequence of V 5), i.e. from 5' to 3': ##STR3## This pool consists of 2⁴ ×4=64 combinations. The probes are labeled with terminal deoxynucleotide transferase (TdT) (marketed by IBI Inc.).

The reaction is carried out on 100 ng of a mixture of oligonucleotides in solution (100 mg/ml) in "Cobalt" reaction buffer (supplied as a 10-fold concentrate by IBI Inc.): 1.4M potassium cacodylate-pH 7.2, 300 mM dithiothreitol, 1 μl of the enzyme terminal deoxynucleotide transferase (IBI Inc.) and 50 μCi of deoxycytidyl triphosphate, dCTP, labeled with P32. The reaction is carried out at 37° C. for 10 min and is then stopped by the addition of 1 μl of EDTA 0.5M. A phenol extraction is carried out and the extract is dialyzed on a column of Biogel P1O polyacrylamide (Biorad: 150-1050).

2) Hybridization and detection of the colonies containing urate oxidase cDNA

About 40,000 colonies are screened by the in situ hybridization technique developed by Grunstein and Hogness (1975, Proc. Natl. Acad. Sci. (U.S.A.), 72, 3961). About 6000 bacteria are plated out in Petri dishes to give isolated colonies. After incubation for 24 h at 37° C., each dish is replicated on 2 filters, each filter being intended to be treated with one of the 2 pools of probes, so that all the colonies obtained are tested with the 2 pools of probes in parallel.

The filters are hybridized with one of the 2 pools of probes in a buffer containing 6×SSC, 10×Denhardt's solution and 100 μg/ml of sonicated and denatured salmon sperm DNA (SIGMA). The hybridization is carried out at a temperature of 42° C. for 16 h. The 6×SSC solution is obtained by diluting a 20×SSC solution. The preparation of the 20×SSC buffer is described by Maniatis, Fritsch and Sambrook (op. cit.). In summary, this buffer contains 175.3 g/l of NaCl and 88.2 g/l of sodium citrate and is adjusted to pH 7 with a few drops of NaOH 10N. The 10×Denhardt's solution contains 1 g of Ficoll, 1 g of polyvinylpyrrolidone and 1 g of human serum albumin per 500 ml of final volume.

After washing in the 6×SSC solution at 42° C. (3 h with 5 changes of bath), the filters are wiped with Joseph paper and subjected to autoradiography. The filters are developed after 16 h. A fraction of about 0.5% of the colonies was found to have hybridized with the 2 pools of probes.

5 colonies from this fraction were taken up and purified. The plasmid DNA was prepared from each of these colonies and this DNA was analyzed by digestion with either BamHI, or HindIII, or both BamHI and HindIII.

After analysis on agarose gel, the 5 plasmids obtained were found to have been linearized by BamHI and by HindIII. The double digestions make it possible to release a fragment corresponding to the whole of the cloned cDNA. The size of this fragment is about 1.2 kb in 3 cases and about 0.9 kb in the other 2 cases. For the following determination, one of the 0.9 kb fragments and one of the 1.2 kb fragments were selected and recloned (see Example 6 below).

EXAMPLE 6: Determination of the sequence of urate oxidase cDNA

On the one hand one of the 0.9 kb fragments (clone 9A) and on the other hand one of the 1.2 kb fragments (clone 9C) were recloned in the DNA of the replicative form of single-stranded phage M13. The DNA of the M13 clones, containing the 0.9 kb fragment on the one hand and the 1.2 kb fragment on the other, was digested with exonuclease so as to generate a series of overlapping M13 clones (procedure: "Cyclone I Biosystem" of IBI). Said clones were sequenced by the dideoxyribonucleotide method (Sanger et al., PNAS-U.S.A.-1977, 14, 5463-5467).

The nucleotide sequence of clone 9C is shown in FIG. 3, which also indicates, with an arrow, the start of clone 9A and, with a nucleotide symbol followed by an asterisk *, the sequenced nucleotides of clone 9A which are not identical to those of clone 9C (when matching the two sequences and the AccI and BamHI restriction sites used in the subsequent constructions (cf. Example 10)).

It is found that

the nucleotide sequence of the longer fragment (clone 9C) overlaps that of the shorter fragment (clone 9A) but for two differences (see FIG. 3). One of the differences is quiescent and the other corresponds to a change from a tryptophan residue to a glycine residue. These differences may be due either to differences in the messenger RNA's isolated (cf. Example 2 above) or to errors in the reverse transcriptase used when building the cDNA library (cf. Example 3 above). The sequencing of the genomic DNA of A. flavus urate oxidase has made it possible to overcome this ambiguity: it is a tryptophan residue (hence probably an error of the reverse transcriptase.

In the case of the longer fragment, an ATG codon (in position 109 in FIG. 3) opens an open reading frame corresponding to a polypeptide of 302 amino acids, with a molecular weight of about 34,240 Da, whose sequence corresponds to the partial sequence of purified A. flavus urate oxidase (cf. Example 4).

FIG. 4 shows the DNA sequence opened by the ATG codon and the polypeptide coded for, and, with arrows opposite the polypeptide coded for, the sequenced peptides (cf. Example 4) obtained by hydrolysis of A. flavus urate oxidase with trypsin and protease V8.

It is found that the sequence of the polypeptide terminates in the triplet Ser-Lys-Leu, which is typical of peroxisomal location enzymes (Gould S. J. et al., J. Cell Biology 108 (1989) 1657-1664).

EXAMPLE 7: Construction of an expression vector for urate oxidase cDNA

Plasmid p466, a vector for expression in E. coli, was prepared. It comprises a fragment of pBR327 including the origin of replication and the ampicillin resistance gene; it also comprises a synthetic promoter of E. coli (R. RODRIGUEZ and M. CHAMBERLIN, "Promoters-Structure and function (1982), Preager), a Shine-Dalgarno sequence followed by a polylinker containing the unique NdeI and KpnI sites, a transcription terminator (derived from phage fd) and the lac i gene.

This plasmid was constructed from an expression plasmid for hGH in E. coli (p462) by replacing a fragment carrying the hGH gene with urate oxidase cDNA.

The construction of plasmid p466 will now be described in greater detail in the following account, which will refer to FIGS. 5, 6, 7, 8 and 9.

FIG. 5 shows a restriction map of plasmid p163,1. The different restriction segments are labeled arbitrarily according to the following legend:

=DNA segment derived from plasmid pBR322

=Location of the origin of replication (ORI)

=DNA segment containing the sequence coding for a natural precursor of hGH

=DNA segment of phage fd containing a transcription terminator

=DNA segment containing a tryptophanlactose UV5 hybrid promoter-operator

=DNA segment coding for β-lactamase (ApR: ampicillin resistance)

FIG. 6 shows the restriction map of plasmid p160, whose PvuI-XhoI-BamHI(1) and PvuI-ORI-BamHI(2) fragments originate respectively from plasmids p163,1 and pBR327 and whose small BamHI(2)-BamHI(1) fragment is fragment 3 described below.

FIG. 7 shows the restriction map of plasmid p373,2. The different restriction segments are labeled arbitrarily according to the following legend:

=PvuI-BamHI sequence derived from plasmid pBR327

=PvuI-XhoI sequence derived from plasmid p163,1

=XhoI-HincII sequence derived from plasmid p163,1 ##STR4## =Fragment 3 described below =DNA segment of phage fd containing a transcription terminator

FIG. 8 shows a restriction map of plasmid p462, the synthetic BglII-HindIII fragment defined below being represented by:

FIG. 9 shows a restriction map of plasmid p466, the NdeI-KpnI fragment, comprising the gene coding for urate oxidase, being represented by:

1) Construction of plasmid p373,2

The strategy employed uses fragments obtained from pre-existing plasmids available to the public, and fragments prepared synthetically by the techniques now in common use. The cloning techniques employed are those described by T. MANIATIS, E. F. FRITSCH and J. SAMBROOK, Cold Spring Harbor Laboratory (1982). The oligonucleotides are synthesized with the aid of a Biosearch 4600 DNA synthesizer.

Plasmid p163,1 (FIG. 5), described in European patent application A-0245138 and deposited in the CNCM under the reference I-530 on Feb. 17, 1986, was digested with the enzymes PvuI and BamHI. This plasmid contains the gene coding for hGH. The PvuI-BamHI fragment-hereafter called fragment 1-containing the site of action of the restriction enzyme XhoI, shown in FIG. 5, was purified.

Likewise, plasmid pBR327, which is well known to those skilled in the art (q.v. SOBERON, X. et al., Gene, 9 (1980) 287-305), was digested with the enzymes PvuI and BamHI. The PvuI-BamHI fragment--hereafter called fragment 2--containing the origin of replication, was purified.

Fragment 3 was then prepared; this is a synthetic BamHI(1)-BamHI(2) fragment containing the lac i gene and its promoter and it has the following sequence (SEQUENCE ID NO. 22), in which the two ends of the strand are identified by the numbers 1 and 2 in order to specify the orientation of the fragment in the plasmids described in FIGS. 6 and 7:

    __________________________________________________________________________     FRAGMENT 3                                                                     __________________________________________________________________________     BamHI(1)                                                                       5'                                                                                  GATCC GCGGAAGCAT                                                                              AAAGTGTAAA                                                                              GCCTGGGGTG                                                                              CCTAATGAGT                                 GAGCTAACTT                                                                              ACATTAATTG                                                                              CGTTGCGCTC                                                                              ACTGCCCGCT                                                                              TTCCAGTCGG                                 GAAACCTGTC                                                                              GTGCCAGCTG                                                                              CATTAATGAA                                                                              TCGGCCAACG                                                                              CGCGGGGAGA                                 GGCGGTTTGC                                                                              GTATTGGGCG                                                                              CCAGGGTGGT                                                                              TTTTCTTTTC                                                                              ACCAGTGAGA                                 CGGGCAACAG                                                                              CTGATTGCCC                                                                              TTCACCGCCT                                                                              GGCCCTGAGA                                                                              GAGTTGCAGC                                 AAGCGGTCCA                                                                              CGCTGGTTTG                                                                              CCCCACCACC                                                                              CGAAAATCCT                                                                              GTTTGATGGT                                 GGTTAACGGC                                                                              GGGATATAAC                                                                              ATGAGCTGTC                                                                              TTCGGTATCG                                                                              TCGTATCCCA                                 CTACCGAGAT                                                                              ATCCGCACCA                                                                              ACGCGCAGCC                                                                              CGGACTCGGT                                                                              AATGGCGCGC                                 ATTGCGCCCA                                                                              GCGCCATCTG                                                                              ATCGTTGGCA                                                                              ACCAGCATCG                                                                              CAGTGGGAAC                                 GATGCCCTCA                                                                              TTCAGCATTT                                                                              GCATGGTTTG                                                                              TTGAAAACCG                                                                              GACATGGCAC                                 TCCAGTCGCC                                                                              TTCCCGTTCC                                                                              GCTATCGGCT                                                                              GAATTTGATT                                                                              GCGAGTGAGA                                 TATTTATGCC                                                                              AGCCAGCCAG                                                                              ACGCAGACGC                                                                              GCCGAGACAG                                                                              AACTTAATGG                                 GCCCGCTAAC                                                                              AGCGCGATTT                                                                              GCTGGTGACC                                                                              CAATGCGACC                                                                              AGATGCTCCA                                 CGCCCAGTCG                                                                              CGTACCGTCT                                                                              TCATGGGAGA                                                                              AAATAATACT                                                                              GTTGATGGGT                                 GTCTGGTCAG                                                                              AGACATCAAG                                                                              AAATAACGCC                                                                              GGAACATTAG                                                                              TGCAGGCAGC                                 TTCCACAGCA                                                                              ATGGCATCCT                                                                              GGTCATCCAG                                                                              CGGATAGTTA                                                                              ATGATCAGCC                                 CACTGACGCG                                                                              TTGCGCGAGA                                                                              AGATTGTGCA                                                                              CCGCCGCTTT                                                                              ACAGGCTTCG                                 ACGCCGCTTC                                                                              GTTCTACCAT                                                                              CGACACCACC                                                                              ACGCTGGCAC                                                                              CCAGTTGATC                                 GGCGCGAGAT                                                                              TTAATCGCCG                                                                              CGACAATTTG                                                                              CGACGGCGCG                                                                              TGCAGGGCCA                                 GACTGGAGGT                                                                              GGCAACGCCA                                                                              ATCAGCAACG                                                                              ACTGTTTGCC                                                                              CGCCAGTTGT                                 TGTGCCACGC                                                                              GGTTGGGAAT                                                                              GTAATTCAGC                                                                              TCCGCCATCG                                                                              CCGCTTCCAC                                 TTTTTCCCGC                                                                              GTTTTCGCAG                                                                              AAACGTGGCT                                                                              GGCCTGGTTC                                                                              ACCACGCGGG                                 AAACGGTCTG                                                                              ATAAEAGACA                                                                              CCGGCATACT                                                                              CTGCGACATC                                                                              GTATAACGTT                                 ACTGGTTTCA                                                                              CATTCACCAC                                                                              CCTGAATTGA                                                                              CTCTCTTCCG                                                                              GGCGCTATCA                                 TGCCATACCG                                                                              CGAAAGGTTT                                                                              TGCGCCATTC                                                                              GATGGTGTCC                                                                              G         3'                                                                   BamHI(2)                                 __________________________________________________________________________

Fragments 1, 2 and 3 were then ligated to give plasmid p160, shown in FIG. 6.

This plasmid was partially digested with the restriction enzymes HincII and PstI. The large HincII-PstI fragment, containing the origin of replication and shown in FIG. 6, was then ligated with fragment 4 (SEQUENCE ID NO. 23), shown below, which is a synthetic DNA fragment carrying a sequence coding for the first 44 amino acids of a natural precursor of hGH and, upstream from this sequence, regulatory signals. ##STR5##

In this fragment, the amino acids are designated by letters according to the following code:

A=Alanine M=Methionine

C=Cysteine N=Asparagine

D=Aspartic acid P=Proline

E=Glutamic acid Q=Glutamine

F=Phenylalanine R=Arginine

G=Glycine S=Serine

H=Histidine T=Threonine

I=Isoleucine V=Valine

K=Lysine W=Tryptophan

L=Leucine Y=Tyrosine

The sequences -35 (TTGCTT) and -10 (TATAAT) of the promoter sequence, and the Shine-Dalgarno sequence well known to those skilled in the art, are successively underlined in this fragment.

Plasmid p380,1 was obtained in this way.

Plasmid p380,1 was then digested with the restriction enzymes ClaI and NdeI so as to remove therefrom the small ClaI-NdeI fragment of fragment 4 above and to replace it with the ClaI-NdeI fragment below (SEQUENCE ID NO. 24): ##STR6##

The resulting plasmid is plasmid p373,2 (FIG. 7).

2) Construction of plasmid p466

Plasmid p373,2 was subjected to a double digestion with the-enzymes BglII and HindIII. The large fragment derived from this digestion was purified and ligated with a synthetic DNA fragment whose sequence, given below (SEQUENCE ID NO. 25), is intended to reconstitute the end of the hGH gene, followed at the 3' end by the KpnI and SnaBI cloning sites. ##STR7##

This fragment comprises the BglII and HindIII sticky ends. The novel plasmid formed in this way, p462 (cf. FIG. 8), thus comprises a KpnI site and an NdeI site, which will be used for cloning the fragment containing urate oxidase cDNA in the expression vector.

The hybrid plasmid derived from pTZ19R, carrying urate oxidase cDNA of about 1.2 kb (clone 9C) (see Example 3), comprises a unique KpnI site. This site is located a few base pairs downstream from the cDNA cloning site. Furthermore, urate oxidase cDNA contains an AccI site situated near the 5' end.

The AccI-KpnI fragment, comprising the greater part of this cDNA, was therefore isolated and purified. Two complementary oligonucleotides were also synthesized, whose sequence, given below (SEQUENCE ID NO. 26): ##STR8## is intended to reconstitute the 5' end of the cDNA. This synthetic fragment obtained in this way has an NdeI end and another AccII end. The fragment and the synthetic sequence were ligated with the expression vector cut by KpnI and by NdeI. This three-fragment ligation makes it possible to obtain the expression vector, called p466, for E. coli urate oxidase (cf. FIG. 9). This plasmid was subjected to a series of enzymatic hydrolyses with restriction enzymes, which made it possible to verify the presence of the expected restriction sites, in particular those carried by the gene coding for urate oxidase.

Plasmid p466 therefore contains, by construction, a gene coding for urate oxidase, having the following sequence (SEQUENCE ID NO. 3): ##STR9## (The nucleotides which are different from the nucleotides of the cDNA isolated from A. flavus are underlined in the above sequence. These differences were introduced into the synthetic AccI-KpnI fragment so as to have, downstream from the ATG, a nucleotide sequence corresponding more closely to those normally encountered in a prokaryotic gene.)

EXAMPLE 8: Expression of urate oxidase cDNA

The E. coli K12 RR1 strain (Bethesda Research Lab. Inc.) was transformed for ampicillin resistance with plasmid p466 and with a negative control plasmid, pBR322. Ampicillin-resistant colonies were obtained in both cases. 1 colony of each type was cultured in a medium (LB+ampicillin 100 μg/ml). After one night at 37° C., with agitation, the two cultures were diluted 100-fold in the medium (LB+ampicillin 100 μg/ml). After culture for 1 h, IPTG (isopropyl-β-D-thiogalactoside) 1 mM is added for 3 h.

Immunodetection of the urate oxidase by Western blot

1) Procedure

An aliquot corresponding to 0.2 ml at OD=1 is taken from the culture medium obtained after induction with IPTG for 3 h. This aliquot is centrifuged and the supernatant is removed. The residue is then subjected to a Western blot--a technique well known to those skilled in the art--which comprises the following steps:

solubilization of the residue by boiling for 10 min in a buffer, called a loading buffer, consisting of Tris-HCl 0.125M pH 6.8, SDS 4%, bromophenol blue 0.002%, glycerol 20%, β-mercaptoethanol 10% (according to the protocol described by LAEMMLI (U. K. LAEMMLI, Nature, 227 (1970) 680-685));

electrophoretic separation of the different proteins contained in the solubilizate, according to the protocol described by LAEMMLI (U. K. LAEMMLI, Nature, 227 (1970) 680-685); and

transfer of said proteins contained in the gel on to a nitrocellulose filter (according to the technique of H. TOWBIN et al., Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354).

Immunodetection, performed according to the technique of BURNETTE (W. W. BURNETTE, Ana. Biochem. 112 (1981) 195-203), involves the following successive operations:

rinsing the nitrocellulose filter for 10 min with a buffer A (Tris-HCl 10 mM, NaCl 170 mM, KCl 1 mM);

bringing the nitrocellulose filter into contact with a buffer B (buffer A with bovine serum albumin added at a rate of 3 g per 100 ml) for 30 min at 37° C.;

bringing the nitrocellulose filter into contact with an immune serum (polyclonal antibodies recognizing A. flavus urate oxidase) for 1 h at 37° C.;

rinsing the nitrocellulose filter with buffer B;

bringing the nitrocellulose filter into contact with a solution of protein G, labeled with iodine 125 at a rate of 0.1 microcurie/ml, for 1 h at 37° C.;

rinsing the filter with buffer A;

drying the filter between two absorbent sheets;

bringing the filter into contact with an X-ray film; and

developing the film.

2) Results

It is found that the strain transformed by plasmid p466 overproduces a protein with an apparent molecular weight of about 33 kDa, which is recognized by antibodies directed against A. flavus urate oxidase and which is absent from the control strain.

EXAMPLE 9: Assay of the urate oxidase activity

An aliquot corresponding to the equivalent of 0.5 ml at OD=1 is taken from the culture medium obtained after induction with IPTG for 3 h under the culture conditions described in the previous Example. This aliquot is centrifuged and the supernatant is removed. The residues are taken up in 1 ml of TEA (triethanolamine) buffer 0.05M pH 8.9. The cell suspension is sonicated twice for 30 s in ice with a W10 ultrasonic sonicator (set to strength 8 and intensity 4). The extracts are centrifuged at 10,000 g for 10 min and the supernatants are used for the assay.

The above operations are carried out for four colonies taken at random from E. coli K12 transformed by plasmid p466 (colonies A₁, B₁, C₁ and D₁) and one colony transformed by plasmid pBR322.

1) Principle

The conversion of uric acid to allantoin is followed by the decrease in absorbance at 292 nm. The reaction is as follows: ##STR10##

2) Reagents

a) TEA 0.05M pH 8.9/EDTA buffer

7.5 g of TEA (reagent for analysis-Prolabo ref. 287.46.266) are dissolved in 400 ml of distilled water;

0.372 g of Complexon III (Merck-ref. 8418) is dissolved in 50 ml of distilled water;

the two solutions are combined and made up to 500 ml (solution 1);

the pH of this solution is adjusted to 8.9 with HCl 0.2N; and

the volume is made up to 1000 ml with distilled water (solution 2).

b) Uric acid stock solution

100 mg of uric acid (Carbiochem-ref. 6671) are dissolved in 50 ml of solution 1;

the pH is adjusted to 8.9 with HCl 0.2N; and

the volume is made up to 100 ml with distilled water.

The solution obtained can be stored for one week at 4° C.

c) Uric acid substrate solution

1.5 ml of uric acid stock solution (Carbiochem-ref. 6671) are taken and diluted to 100 ml with TEA buffer (reagent for analysis-Prolabo ref. 287.46.266).

This solution must be used the same day.

3) Procedure

The following volumes are introduced into the quartz cell of a spectrophotometer set to 292 nm and thermostated at 30° C.:

600 μl of uric acid substrate solution (preheated to 30° C.) and

100 μl of the above supernatants to which 200 μl of TEA pH 8.9 have been added (preheated to 30° C.).

After mixing, the change in optical density is read off every 30 s for 5 min. ΔE, the variation in optical density per minute, is deduced from these readings.

4) Results

The urate oxidase enzymatic activity A, expressed in U/ml OD 1, is calculated from the ΔE measurement with the aid of the formula ##EQU1## in which the symbols Vr, d, εI and V_(PE) respectively represent the reaction volume (0.9 ml), the dilution factor (2), the extinction coefficient of uric acid at 292 nm (12.5) and the volume of the test sample (0.1 ml).

The results obtained are collated in Table II below:

                  TABLE II                                                         ______________________________________                                         E. coli K12 strain                                                                              Urate oxidase activity                                        transformed by   (U/ml OD 1)                                                   ______________________________________                                         pBR322               <0.001                                                               colony A.sub.1                                                                           0.086                                                                colony B.sub.1                                                                           0.119                                                     p466                                                                                      colony C.sub.1                                                                           0.135                                                                colony D.sub.1                                                                           0.118                                                     ______________________________________                                    

The above Table clearly shows that the E. coli cells transformed by plasmid p466 are capable of producing urate oxidase activity in the presence of IPTG.

EXAMPLE 10: Construction of three expression vectors for urate oxidase cDNA in yeast: plasmids pEMR469, pEMR473 and pEMR515

The strategy employed uses fragments obtained from pre-existing plasmids available to the public, and fragments prepared synthetically by the techniques now in common use. The cloning techniques employed are those described by T. MANIATIS, E. F. FRITSCH and J. SAMBROOK in "Molecular Cloning, a laboratory manual" (Cold Spring Harbor Laboratory, 1984). The oligonucleotides are synthesized with the aid of a Biosearch 4600 DNA synthesizer.

The following description will be understood more clearly with reference to FIGS. 10, 11 and 12, which respectively show restriction maps of plasmids pEMR414, pEMR469 and pEMR473. The symbols used in these Figures will be specified in the description below. In the case where a site has been blunted by Klenow polymerase, it carries the index "°"; where the sites have been eliminated by ligation, they are indicated in brackets.

1) Construction of plasmid pEMR469

This plasmid was constructed from the shuttle vector E. coli-yeast pEMR414, constructed by successive ligations of the following components:

the PstI-HindIII° fragment--symbolized by in FIG. 10--of plasmid pJDB207 (BEGGS, 1978: Gene cloning in yeast---p. 175-203 in: Genetic Engineering, vol. 2-WILLIAMSON-Academic Press-London UK) comprising the upstream part of the ampicillin resistance gene Amp^(R) of pBR322 (Sutcliffe, 1979, Cold Spring Symp. Quart. Biol. 43, 779) and an endogenous 2μ fragment, B form, carrying the LEU2 gene of S. cerevisiae partially modified by the deletion of its promoter (called LEU2d), the locus STB (REP3) and the origin of replication of the 2μ fragment (HARTLEY and DONELSON, 1980, Nature, 286, 860-865). The HindIII end of this fragment has been blunted by the action of Klenow polymerase. It is denoted by HindIII° in FIG. 10.

the HindIII-SmaI fragment--represented by in FIG. 10--of chromosome V of yeast containing the URA3 gene with its promoter (ROSE et al., 1984, Gene, 29, p. 113-124). This HindIII-SmaI fragment originates from plasmid pFL1 (CHEVALLIER et al., 1980, Gene 11, 11-19). The HindIII end of this plasmid has been blunted by the action of Klenow polymerase.

an SamI-BamHI fragment--symbolized by in FIG. 10--containing a synthetic version of the promoter of the ADH2 gene which differs from the natural version described by RUSSEL and SMITH (RUSSEL et al. (1983) J. Biol. Chem. 258, 2674-2682) only by a few base pairs intended for introducing restriction sites. (The natural sequence could be used with only slightly different results.) The sequence of this fragment is given below (SEQUENCE ID NO. 28): ##STR11## the BgIII-HindIII fragment--symbolized by in FIG. 10--carrying the 3' end of the yeast PGK gene. This fragment originates from complete digestion with BgIII of the HindIII fragment of the yeast chromosomal DNA, carrying the PGK gene described by HITZEMAN et al. (1982, Nucleic Acids Res., 10, 7791-7808), which has only one BgIII site. This digestion makes it possible to obtain two HindIII-BgIII fragments of which the smaller, of about 0.4 kb, which carries the 3' end of the yeast PGK gene, is retained. The sequence of the latter fragment is described by HITZEMANN et al. (op. cit.). The BgIII site is cloned in the BamHI site of the previous fragment (the BamHI and BgIII sites therefore disappearing), and the HindIII site, blunted by the action of Klenow polymerase, is cloned in the PvuII site of the PvuII-PstI fragment of pBR322, described below.

the PvuII-PstI fragment--symbolized by in FIG. 10--of pBR322, containing the origin of replication and the downstream part of the ampicillin resistance gene Amp^(R).

Plasmid pEMR414 formed in this way therefore contains the following components:

an origin of replication and an ampicillin resistance gene Amp^(R) permitting the replication and selection of the plasmid in E. coli cells. These components permit transformation in E. coli cells.

an origin of replication for the yeast (ARS), the locus STB and the LEU2 gene of S. cerevisiae without promoter and the URA3 gene of S. cerevisiae with its promoter. These components permit the replication and selection of the plasmid in S. cerevisiae cells and a sufficient partition efficacy in cells containing the endogenous 2μ plasmid.

Plasmid pEMR414 was completely digested with the restriction enzymes NheI and ClaI. The small NheI-ClaI fragment containing the URA3 gene, hereafter called fragment A, was purified.

Plasmid pEMR414 was completely digested with the enzymes NheI and BamHI. The large NheI-BamHI fragment containing especially the LEU2d gene and the origin of replication of plasmid pBR322, hereafter called fragment B, was purified.

The synthetic ClaI-AccI fragment, containing the start of a gene coding for the protein deduced from the urate oxidase cDNA sequence (clone 9C), was also prepared. This fragment contains modifications, relative to clone 9C, introduced for the purpose of inserting codons which are customary in yeast (q.v. SHARP et al., 1986, Nucl. Ac. Res., vol. 14, 13, pp. 5125-5143) without changing the amino acids coded for. The sequence of this fragment, hereafter called fragment C, is as follows (SEQUENCE ID NO. 29)(the underlined nucleotides are those modified relative to clone 9C): ##STR12##

The plasmid of clone 9C. (cf. FIG. 3) was digested with the enzymes AccI and BamHI. The AccI-BamHI fragment, which contains the end of urate oxidase cDNA, hereafter called fragment D, was purified. This fragment has the following sequence (SEQUENCE ID NO. 30): ##STR13##

Fragments A, B, C and D were ligated to give plasmid pEMR469 shown in FIG. 11, in which the symbols have the same meanings as in FIG. 10, the novel ClaI-AccI and AccI-BamHI fragments being symbolized by .

2) Construction of plasmid pEMR473

Plasmid pEMR469 was completely digested with the enzymes MluI and SphI. The large MluI-SphI fragment, containing the urate oxidase gene, was then ligated with the synthetic fragment, whose sequence is given below, corresponding to a part (200 bp) of the sequence upstream from the TATA component of promoter GAL7 of S. cerevisiae, said part comprising the upstream activation sequences (UAS). ##STR14##

Plasmid pEMR473 obtained in this way is shown in FIG. 12, in which the symbols have the same meanings as in FIG. 11, the novel MluI-SphI fragment introduced being symbolized by .

3) Construction of plasmid pEMR515

Plasmid pEMR473 was partially digested with the enzyme XbaI and totally digested with the enzyme MluI. The large XbaI-MluI fragment was purified. This fragment contains especially the sequences of the origin of replication and the locus STB of the 2μ fragment, the LEU2d gene, the ampicillin resistance gene Amp^(R), the origin of replication of pBR322 and the expression cassette for urate oxidase. On the other hand, it contains neither the URA3 gene nor that part of the 2μ fragment which is between the XbaI and NheI sites.

The large XbaI-MluI fragment was recircularized via the following sequence adapter (SEQUENCE ID NO. 32) containing MluI and modified XbaI sticky ends: ##STR15##

Plasmid pEMR515 obtained in this way has only one of the three components of the target FRT site of the recombinase coded for by the FLP gene of the 2μ fragment.

Plasmids pEMR469, pEMR473 and pEMR515 possess the gene coding for urate oxidase, which has the following sequence (SEQUENCE ID NO. 4):

    __________________________________________________________________________     ATGTCTGCTG                                                                              TTAAGGCTGC                                                                              TAGATACGGT                                                                              AAGGACAACG                                                                              TTAGAGTCTA                                 CAAGGTTCAC                                                                              AAGGACGAGA                                                                              AGACCGGTGT                                                                              CCAGACGGTG                                                                              TACGAGATGA                                 CCGTCTGTGT                                                                              GCTTCTGGAG                                                                              GGTGAGATTG                                                                              AGACCTCTTA                                                                              CACCAAGGCC                                 GACAACAGCG                                                                              TCATTGTCGC                                                                              AACCGACTCC                                                                              ATTAAGAACA                                                                              CCATTTACAT                                 CACCGCCAAG                                                                              CAGAACCCCG                                                                              TTACTCCTCC                                                                              CGAGCTGTTC                                                                              GGCTCCATCC                                 TGGGCACACA                                                                              CTTCATTGAG                                                                              AAGTACAACC                                                                              ACATCCATGC                                                                              CGCTCACGTC                                 AACATTGTCT                                                                              GCCACCGCTG                                                                              GACCCGGATG                                                                              GACATTGACG                                                                              GCAAGCCACA                                 CCCTCACTCC                                                                              TTCATCCGCG                                                                              ACAGCGAGGA                                                                              GAAGCGGAAT                                                                              GTGCAGGTGG                                 ACGTGGTCGA                                                                              GGGCAAGGGC                                                                              ATCGATATCA                                                                              AGTCGTCTCT                                                                              GTCCGGCCTG                                 ACCGTGCTGA                                                                              AGAGCACCAA                                                                              CTCGCAGTTC                                                                              TGGGGCTTCC                                                                              TGCGTGACGA                                 GTACACCACA                                                                              CTTAAGGAGA                                                                              CCTGGGACCG                                                                              TATCCTGAGC                                                                              ACCGACGTCG                                 ATGCCACTTG                                                                              GCAGTGGAAG                                                                              AATTTCAGTG                                                                              GACTCCAGGA                                                                              GGTCCGCTCG                                 CACGTGCCTA                                                                              AGTTCGATGC                                                                              TACCTGGGCC                                                                              ACTGCTCGCG                                                                              AGGTCACTCT                                 GAAGACTTTT                                                                              GCTGAAGATA                                                                              ACAGTGCCAG                                                                              CGTGCAGGCC                                                                              ACTATGTACA                                 AGATGGCAGA                                                                              GCAAATCCTG                                                                              GCGCGCCAGC                                                                              AGCTGATCGA                                                                              GACTGTCGAG                                 TACTCGTTGC                                                                              CTAACAAGCA                                                                              CTATTTCGAA                                                                              ATCGACCTGA                                                                              GCTGGCACAA                                 GGGCCTCCAA                                                                              AACACCGGCA                                                                              AGAACGCCGA                                                                              GGTCTTCGCT                                                                              CCTCAGTCGG                                 ACCCCAACGG                                                                              TCTGATCAAG                                                                              TGTACCGTCG                                                                              GCCGGTCCTC                                                                              TCTGAAGTCT                                 AAATTG.                                                                        __________________________________________________________________________

EXAMPLE 11: Transformation of the EMY761 yeast strain by plasmids pEMR469, pEMR473 and pEMR515-Transformation of the EMY500 and GRF18 yeast strains by plasmid pEMR515-Transformation with selection either for the prototrophy of uracil or for the prototrophy of leucine

Three non-isogenic strains of Saccharomyces cerevisiae were used as recipient strains:

the EMY761 strain (Matα, leu2, ura3, his3, gal)

the EMY500 strain (Matα, leu2, ura3, pep4)

the GRF18 strain (Matα, leu2, his3)

The GRF18 strain is well known to those skilled in the art (Gerry FINK, MIT, U.S.A.). The EMY761 and EMY500 strains are related to the GRF18 strain. They were obtained by successively crossing the GRF18 strain with a ura3 strain derived from the FL100 strain (deposited in the ATCC under n° 28 383) and with the 20B12 strain (Matα, tsp1, pep4) described by E. W. JONES (E. W. JONES et al. (1977) Genetics, 85, 23).

The GRF18 strain can be obtained by curing plasmid pEMR515 of the GRF18 pEMR515 (leu⁺) strain deposited in the CNCM under reference n° I-920 on Dec. 28, 1989, and the EMY500 strain can be obtained by curing plasmid pEMR515 of the EMY500 pEMR515 (leu⁺) strain deposited in the CNCM under reference n° I-919 on Dec. 28, 1989.

These strains contain mutations (leu2 and ura3 capable of being complemented by the LEU2d defective selection marker and the URA3 selection marker, which are present in each of plasmids pEMR469 and pEMR473.

1) Transformation with selection for the prototrophy of uracil

A colony of the EMY761 strain was used to inoculate 100 ml of a medium called liquid YPG medium (cf. Table III below). When the cell density had reached 10⁷ cells per ml, the cells were treated with lithium acetate 0.2M for transformation by a technique well known to those skilled in the art and described by ITO et al. (ITO et al., 1983, J. Bacteriology 153, 163-168).

The EMY761 cells were transformed in parallel with about 1 μg of each of plasmids pEMR469 and pEMR473. The transformed cells are selected for the auxotrophic character of uracil (ura⁺) on a medium called uracil-free solid medium (cf. Table III below). An EMY761 pEMR469 (ura⁺) transformed strain and an EMY761 pEMR473 (ura⁺) transformed strain were thus retained.

2) Transformation with selection for the prototrophy of leucine

The transformation technique used is a variant of that described by Beggs et al. (Beggs et al. (1978) Nature 275, 104-109). It consists in subjecting yeasts to a protoplastization treatment in the presence of an osmotic stabilizer, namely sorbitol at a concentration of 1M.

The precise transformation protocol is specified below:

a) 200 ml of liquid YPG medium (cf. Table III) are inoculated with about 5×10⁶ cells of a culture in the stationary phase, and the culture inoculated in this way is agitated overnight at 30° C.

b) When the density of the culture reaches about 10⁷ cells per ml, the cells are centrifuged at 4000 rpm for 5 min and the residue is washed with sorbitol 1M.

c) The cells are suspended in 5 ml of sorbitol solution 1M containing 25 mM EDTA and 50 mM dithiothreitol, and are incubated for 10 min at 30° C.

d) The cells are washed once with 10 ml of sorbitol 1M and suspended in 20 ml of sorbitol. Zymolase-100T (a preparation obtained by partial purification of Arthobacter luteus culture supernatant on an affinity column and containing β-1,3-glucan laminaripentahydrolase, marketed by SEYKAGAKU KOGYO Co. Ltd.) is added up to a final concentration of 20 μg/ml and the suspension is incubated at room temperature for about 15 min.

e) The cells are resuspended in 20 ml of a medium containing sorbitol, called sorbitol YPG medium (cf. Table III below) and incubated for 20 min at 30° C., with gentle agitation.

f) The cells are centrifuged for 3 min at 2500 rpm.

g) The cells are resuspended in 9 ml of transformation formation buffer (sorbitol 1M, Tris-HCl 10 mM pH 7.5 and CaCl₂ 10 mM).

h) 0.1 ml of cells and 5 μl of DNA solution (about 5 μg) are added and the suspension obtained is left for 10 to 15 min at room temperature.

i) 1 ml of the following solution is added: polyethylene glycol PEG 4000 20%, Tris-HCl 10 mM pH 7.5 and CaCl₂ 10 mM.

j) 0.1 ml of the suspension obtained in i) is poured into a tube containing leucine-free solid regeneration medium (cf. Table III below) which has been melted beforehand and kept liquid at about 45° C. The suspension is poured into a Petri dish containing a solidified layer of 15 ml of leucine-free solid regeneration medium.

k) Step j) is repeated with the remainder of the cell suspension obtained in i).

The transformed strains start to appear after three days.

The EMY761 pEMR469 (leu⁺), EMY761 pEMR473 (leu⁺), EMY761 pEMR515 (leu⁺), GRF18 pEMR515 (leu⁺) and EMY500 pEMR515 (leu⁺) transformed strains were thus retained.

    ______________________________________                                         Principal media used in Examples 11, 12, 13 and 14                             ______________________________________                                         uracil-free solid medium                                                       6.7 g of Yeast nitrogen base without Amino Acids (from                         DIFCO)                                                                         5.0 g of casein hydrolyzate (Casamino acids from DIFCO)                        10 g of glucose                                                                20 g of agar                                                                   Mix all the ingredients in distilled water and make up                         the final volume to 1 l with distilled water. Auto-                            clave for 15 min at 120° C.                                             uracil-free liquid medium                                                      Use the formulation of the uracil-free solid medium                            without the agar. Autoclave for 15 min at 120° C.                       leucine-free solid medium                                                      6.7 g of Yeast nitrogen base without Amino Acids (from                         DIFCO)                                                                         20 mg of adenine                                                               20 mg of uracil                                                                20 mg of l-tryptophan                                                          20 mg of l-histidine                                                           20 mg of l-arginine                                                            20 mg of l-methionine                                                          30 mg of l-tyrosine                                                            30 mg of l-isoleucine                                                          30 mg of l-lysine                                                              50 mg of l-phenylalanine                                                       100 mg of l-glutamic acid                                                      150 mg of l-valine                                                             400 mg of l-leucine                                                            20 g of glucose                                                                20 g of agar                                                                   Mix all the ingredients in distilled water. Make up                            the final volume to 1 l with distilled water. Auto-                            clave for 15 min at 120°  C. After autoclaving, add 200                 mg of l-threonine and 100 mg of 1-aspartic acid-                               leucine-free solid regeneration medium                                         Use the formulation of the leucine-free solid medium,                          mixing in 30 g of agar instead of 20 g and adding 182 g                        of sorbitol to the mixture.                                                    leucine-free liquid medium                                                     Use the formulation of the leucine-free solid medium                           without the agar. Autoclave for 15 min at 120° C.                       After autoclaving, add 200 mg of l-threonine and 100 mg                        of l-aspartic acid.                                                            liquid YP medium                                                               10 g of yeast extract (Bacto-yeast extract from DIFCO)                         20 g of peptone (Bacto-peptone from DIFCO)                                     Mix the ingredients in distilled water. Make up the                            final volume to 1 l with distilled water. Autoclave                            for 15 min at 120° C.                                                   liquid YPG medium                                                              Use the formulation of the liquid YP medium, adding,                           after autoclaving, glucose at a concentration of 20                            g/l.                                                                           sorbitol YPG medium                                                            Use the formulation of the liquid YPG medium, adding,                          after autoclaving, sorbitol at a concentration of 1 M.                         ethanol-glycerol YP medium                                                     Use the formulation of the liquid YP medium. After                             autoclaving, add 10 ml of ethanol 100% (1% final con-                          centration) and 30 g of glycerol.                                              ethanol-glycerol-galactose YP medium                                           Use the formulation of the liquid YP medium. After                             autoclaving, add 10 ml of ethanol 100%, 30 g of                                glycerol and 30 g of galactose.                                                ______________________________________                                    

EXAMPLE 12: Expression, in an Erlenmeyer flask, of urate oxidase cDNA by the EMY761 pEMR469 (ura⁺), EMY761 pEMR473 (ura⁺), EMY761 pEMR469 (leu⁺) and EMY761 pEMR473 (leu⁺) strains-Immunodetection by Western blot-Assay of the urate oxidase activity and the soluble proteins 1) Expression of urate oxidase cDNA

a) Strains selected on uracil-free medium

A colony of each of the EMY761 pEMR469 (ura⁺) and EMY761 pEMR473 (ura⁺) strains was cultured in 20 ml of uracil-free liquid medium (cf. Table III, Example 11). After one night at 30° C., with agitation, the two cultures were centrifuged for 10 min at 7000 rpm. The residues were taken up in 10 ml of sterile distilled water and centrifuged again for 10 min at 7000 rpm. Expression of the urate oxidase was induced by taking up the cells in 20 ml of ethanol-glycerol YP medium (cf. Table III, Example 11) for the EMY761 pEMR469 (ura⁺) strain and in 20 ml of ethanol-glycerol-galactose YP medium (cf. Table III, Example 11) for the EMY761 pEMR473 (ura⁺) strain. The cultures were incubated again at 30° C. for 22 h, with agitation.

b) Strains selected on leucine-free medium

In a first stage, a colony of each of the EMY761 pEMR469 (leu⁺) and EMY761 pEMR473 (leu⁺) strains was cultured in 20 ml of leucine-free liquid medium (cf. Table III, Example 11). This made it possible to obtain and maintain a large number of copies of plasmids by carrying out the selection for complementation of the leu2mutation by the LEU2d gene carried by plasmids pEMR469 and pEMR473.

After one night at 30° C., with agitation, the two cultures were centrifuged for 10 min at 7000 rpm. The residues were taken up in 10 ml of sterile distilled water and centrifuged again for 10 min at 7000 rpm. Expression of the urate oxidase was induced by taking up the cells in 20 ml of ethanol-glycerol YP medium for the EMY761 pEMR469 (leu⁺) strain and in 20 ml of ethanol-glycerol-galactose YP medium (cf. Table III, Example 11) for the EMY761 pEMR473 (leu⁺) strain. The cultures were incubated again at 30° C. for 22 h, with agitation.

c) Control strain

The non-transformed EMY761 strain, i.e. the EMY761 strain without plasmid, was cultivated as above. It was subjected on the one hand to induction in 10 ml of ethanol-glycerol liquid YP medium and on the other hand to induction in 10 ml of ethanol-glycerol-galactose YP medium.

2) Preparation of the samples

a) The cells cultivated in 1a), 1b) and 1c) were centrifuged and the supernatant was removed. The residues were taken up in 10 ml of distilled water and centrifuged for 10 min at 7000 rpm. The residues washed in this way were taken up in about 1 ml of triethyleneamine buffer, TEA, of pH 8.9. About 300 μl of cells taken up in said buffer were lyzed in the presence of glass beads (from 400 to 500 μm in diameter), representing about half the final volume. This mixture was agitated vigorously in a Vortex 4 times for 1 min, the samples being placed in ice for 30 s between grinding operations. The liquid was withdrawn from the tubes with a Pasteur pipette and transferred to a microtube. The glass beads were washed once with about 200 μl of TEA buffer of pH 8.9. The beads were agitated in a Vortex once for 1 min and the liquid was withdrawn with a Pasteur pipette and added to the above lyzate. The lyzate was then centrifuged in a microtube for 5 min at 7000 rpm. The supernatant was cautiously withdrawn and stored at -20° C. for Western blot, assay of the urate oxidase activity and assay of the proteins. The residue of the lyzed cells was stored separately at -20° C. for Western blot (cf. 3) below).

Furthermore, samples of the cultures prepared in 1a) and 1b) were taken in the following manner before induction: 2 ml of culture were centrifuged for 10 min at 7000 rpm. The residues were taken up in 500 μl of distilled water and centrifuged again for 5 min at 7000 rpm. The residues were taken up in about 200 μl of TEA buffer of pH 8.9 and lyzed as above in the presence of glass beads. The supernatants and the residues of the lyzed cells were stored separately at -20° C.

3) Immunodetection of the urate oxidase by Western blot

a) Procedure

The residues and the supernatants of the different samples were subjected to a Western blot--a technique well known to those skilled in the art--which comprises the following steps:

solubilization of the residue by boiling for 10 min in a buffer, called a loading buffer, consisting of Tris-HCl 0.125M pH 6.8, SDS 4%, bromophenol blue 0.002%, glycerol 20%, β-mercaptoethanol 10% (according to the protocol described by LAEMMLI (U. K. LAEMMLI, Nature, 227 (1970) 680-685));

electrophoretic separation of the different proteins contained in the solubilizate, according to the protocol described by LAEMMLI (U. K. LAEMMLI, Nature, 227 (1970) 680-685); and

transfer of said proteins contained in the gel on to a nitrocellulose filter (according to the technique of H. TOWBIN et al., Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354).

Immunodetection, performed according to the technique of BURNETTE (W. W. BURNETTE, Ana. Biochem. 112 (1981) 195-203), involves the following successive operations:

rinsing the nitrocellulose filter for 10 min with a buffer A (Tris-HCl 10 mM, NaCl 170 mM, KCl 1 mM);

bringing the nitrocellulose filter into contact with a buffer B (buffer A with bovine serum albumin added at a rate of 3 g per 100 ml) for 30 min at 37° C.;

bringing the nitrocellulose filter into contact with an immune serum (polyclonal antibodies recognizing A. flavus urate oxidase) for 1 h at 37° C.;

rinsing the nitrocellulose filter with buffer B;

bringing the nitrocellulose filter into contact with a solution of protein G, labeled with iodine 125 at a rate of 0.1 microcurie/ml, for 1 h at 37° C.;

rinsing the filter with buffer A;

drying the filter between two absorbent sheets;

bringing the filter into contact with an X-ray film; and

developing the film.

b) Results

It is found that the EMY761 pEMR469 (ura⁺), EMY761 pEMR473 (ura⁺), EMY761 pEMR469 (leu⁺) and EMY761 pEMR473 (leu⁺) strains produce a protein with an apparent molecular weight of about 33 kDa, which is recognized by antibodies directed against A. flavus urate oxidase and which is absent from the control strain.

It is also found that the non-induced strains produce none or very little of the protein described above.

Comparison between the amounts of this protein for the residues and the supernatants makes it possible to deduce that about 80% of said protein is in soluble form in the lyzate.

4) Assay of the urate oxidase activity

The urate oxidase activity was measured on the supernatants of the lyzed cells according to the procedure described in Example 9 above.

The results obtained are collated in Table IV below, which specifies the urate oxidase activity in U/ml for each strain induced by glycerol-ethanol, each strain induced by glycerol-ethanol-galactose and each non-induced strain.

                  TABLE IV                                                         ______________________________________                                                                  Urate oxidase                                                                  activity                                              Strain/Inducer           (U/ml)                                                ______________________________________                                         EMY761/YP ethanol-glycerol-galactose                                                                    <0.1                                                  EMY761/YP ethanol-glycerol                                                                              <0.1                                                  EMY761 pEMR469 (ura.sup.+)/(non-induced)                                                                0.4                                                   EMY761 pEMR469 (ura.sup.+)/YP ethanol-glycerol                                                          12                                                    EMY761 pEMR469 (leu.sup.+)/(non-induced)                                                                0.17                                                  EMY761 pEMR469 (leu.sup.+)/YP ethanol-glycerol                                                          36                                                    EMY761 pEMR473 (ura.sup.+)/(non-induced)                                                                <0.1                                                  EMY761 pEMR473 (ura.sup.+)/YP ethanol-glycerol-                                                         12.5                                                  galactose                                                                      EMY761 pEMR473 (leu.sup.+)/(non-induced)                                                                <0.1                                                  EMY761 pEM473 (leu.sup.+)/YP ethanol-glycerol-                                                          15.3                                                  galactose                                                                      ______________________________________                                    

The above Table clearly shows that the yeast cells transformed by these plasmids pEMR469 and pEMR473 are capable of producing urate oxidase activity after induction.

5) Assay of the total soluble proteins in the lyzates

The protein assay kit from BIORAD was used for assaying the total proteins present in the supernatant of the lyzed cells. It is based on the observation that the maximum absorbance of an acid solution of Coomassie brilliant blue g-250 changes from 465 nm to 595 nm when proteins become attached thereto (q.v. Reisner et al., Anal. Biochem., 64, 509 (1975)).

a) Procedure

The following volumes are introduced into the cell of a spectrophotometer set to 595 nm:

10 μl of sample to which 790 μl of distilled water have been added

200 μl of concentrated Dye reagent (Biorad).

The ingredients are mixed and the optical density is read off at 595 nm. A calibration range with increasing concentrations of BSA (bovine serum albumin) was prepared in this way. The unknown concentration of the total proteins in the lyzates is read off on the calibration curve obtained.

b) Results

The main results obtained are collated in Table V below, which specifies the mount (in mg/ml) of total soluble proteins and the percentage of urate oxidase in the total soluble proteins for each strain induced by glycerol-ethanol, each strain induced by glycerol-ethanol-galactose and each non-induced strain (it is assumed here that the specific activity of the recombinant protein is identical to that of the urate oxidase obtained from A. flavus: 30 U/mg).

                  TABLE V                                                          ______________________________________                                                              Total    % of urate                                                            soluble  oxidase in the                                                        proteins total soluble                                    Strain/Inducer       mg/ml    proteins                                         ______________________________________                                         EMY761/glycerol-ethanol                                                                             5.3      <0.05                                            EMY761/glycerol-ethanol-galactose                                                                   5.8      <0.05                                            EMY761 pEMR469 (ura.sup.+)/non-induced                                                              8.5      0.25                                             EMY761 pEMR469 (ura.sup.+)/glycerol-                                                                5.3      4.7                                              ethanol                                                                        EMY761 pEMR469 (leu.sup.+)/non-induced                                                              1.7      0.3                                              EMY761 pEMR469 (leu.sup.+)/glycerol-                                                                5.9      20                                               ethanol                                                                        EMY761 pEMR473 (ura.sup.+)/non-induced                                                              10.3     <0.05                                            EMY761 pEMR473 (ura.sup.+)/glycerol-                                                                6.5      6.4                                              ethanol galactose                                                              EMY761 pEMR473 (leu.sup.+)/non-induced                                                              0.5      <0.05                                            EMY761 pEMR473 (leu.sup.+)/glycerol-                                                                3.9      13                                               ethanol-galactose                                                              ______________________________________                                    

It is found that the production rate of urate oxidase varies from 5 to 20% according to the transformants and the mode of selection of the transformed strains (leu⁺).

EXAMPLE 13: Expression, in a 2.5 l fermenter, of urate oxidase cDNA by the EMY761 pEMR473 (ura⁺) strain 1) Fermentation protocol

a) Media

Inoculum medium

A colony of the EMY761 pEMR473 (ura⁺) strain was cultured in 200 ml of uracil-free liquid medium (cf. Table III, Example 11). Culture is continued overnight, with agitation, until the OD is about 3.

    ______________________________________                                         Culture medium A                                                                                  for 1 l of purified water                                                      on an apparatus of the                                                         Milli-Q type                                                ______________________________________                                         glucose              30        g                                               glycerol             30        g                                               casein hydrolyzate (Casamino                                                                        30        g                                               acids from DIFCO)                                                              Yeast Nitrogen Base (from DIFCO)                                                                    15        g                                               Yeast extract (from DIFCO)                                                                          2.5       g                                               K.sub.2 HPO.sub.4    3         g                                               MgSO.sub.4.7H.sub.2 O                                                                               0.5       g                                               ______________________________________                                    

    ______________________________________                                         Additional medium B                                                                             for 100 ml of purified water                                                   on an apparatus of the                                                         Milli-Q type                                                  ______________________________________                                         glycerol           30         g                                                peptone hydrolyzate                                                                               30         g                                                (Primatone from G. Sheffield)                                                  Yeast Nitrogen Base (from                                                                         15         g                                                DIFCO)                                                                         Yeast extract (from DIFCO)                                                                        5          g                                                K.sub.2 HPO.sub.4  3          g                                                MgSO.sub.4.7H.sub.2 O                                                                             0.5        g                                                ______________________________________                                    

b) Fermentation parameters

Bioreactor of total volume 2.5 l, equipped with two turbines

Temperature=30° C.

pH=5

Oxygen partial pressure=30 mm Hg

Air flow rate=1l/min

The bioreactor is filled with 1.5 l of medium A and inoculated with 150 ml of the inoculum.

Once the glucose has been exhausted at OD 2.5 to about OD 17, induction is effected by the addition of a volume of 150 ml of galactose at 20% weight/volume. Growth is continued and additional medium B is then added at about OD 30.

Growth continues for about another fifteen hours and the product was harvested at OD 104.

2) Preparation and analysis of the samples

The samples were prepared as described in Example 9 2) a) from the culture in the fermenter. Two samples were taken: the first after 7 h of induction and the second after 22 h of induction.

The following tests, described in Example 9, were performed on these two lyzates obtained after lysis of the cells:

immunodetection by Western blot

assay of the biological activity

assay of the total proteins

The following results were obtained:

a) Immunodetection by Western blot

It is found that the EMY761 pEMR473 (ura⁺) strain, cultivated in a 2 l fermenter, produces a protein with an apparent molecular weight of 33 kDa, which is recognized by antibodies directed against A. flavus urate oxidase (said antibodies being prepared in rabbits by techniques well known to those skilled in the art: q.v. VAITUKAITIS et al. (1981) "Methods in Enzymology", Academic Press, New York, vol. 73, p. 46) and which is absent from the control strain.

b) Assay of the biological activity

The results obtained are collated in Table VI below:

                  TABLE VI                                                         ______________________________________                                         Strain/Induction time U/ml                                                     ______________________________________                                         EMY761 pEMR473 (ura.sup.+)/7 h                                                                       9                                                        EMY761 pEMR473 (ura.sup.+)/22 h                                                                      12.5                                                     ______________________________________                                    

It is found that the EMY761 pEMR473 (ura⁺) strain, cultivated in a fermenter, is capable of producing urate oxidase activity after induction.

c) Assay of the total soluble proteins

The results are collated in Table VII below:

                  TABLE VII                                                        ______________________________________                                                            Total    % of urate                                                            soluble  oxidase in the                                                        proteins total soluble                                      Strain/Induction time                                                                             mg/ml    proteins                                           ______________________________________                                         EMY761 pEMR473 (ura.sup.+)/7 h                                                                    5.2      5.7                                                EMY761 pEMR473 (ura.sup.+)/21 h                                                                   6.2      6.6                                                ______________________________________                                    

These results indicate that the rate of synthesis of urate oxidase by the EMY761 pEMR473 (ura⁺) strain, cultivated in a fermenter, is about 5% of the total proteins of the cell after 7 h and 21 h of induction.

EXAMPLE 14: Expression, in an Erlenmeyer flask, of urate oxidase cDNA by the EMY761 pEMR515 (leu⁺), EMY500 pEMR515 (leu⁺) and GRF18 pEMR515 (leu⁺) strains

A colony of each of the above three strains was cultured in 20 ml of leucine-free liquid medium.

After one night at 30° C. with agitation the three cultures were centrifuged for 10 min at 7000 rpm. The cell residues were taken up in 10 ml of sterile distilled water and centrifuged again for 10 min. Expression of the urate oxidase was induced by taking up the cells in 20 ml of ethanol-glycerol-galactose YP medium (cf. Table I, Example 8). The cultures were incubated again at 30° C. for about 20 h, with agitation. The non-transformed host strains were each cultured as controls.

The cells of each of the six cultures are separated out again by centrifugation and the supernatant is removed. The residues were taken up in 10 ml of distilled water and centrifuged for 10 min at 7000 rpm. The residues washed in this way were taken up in about 1 ml of TEA buffer of pH 8.9 and the grinding and removal of the particles by centrifugation were carried out as described in Example 9, 2). The supernatant of each culture is used, as previously, for assaying the urate oxidase and the total proteins. The main results obtained are collated in Table VIII below:

                  TABLE VIII                                                       ______________________________________                                                                           % of urate                                               Urate oxidase                                                                             Total soluble                                                                             oxidase in                                   Strain/Culture                                                                             activity   proteins   the soluble                                  conditions  (U/ml)     (mg/ml)    proteins                                     ______________________________________                                         GRF18 pEMR15                                                                               <0.1       2.2        <0.05                                        (leu.sup.+)/a)                                                                 EMY500 pEMR15                                                                              <0.1       0.9        <0.05                                        (leu.sup.+)/a)                                                                 EMY761 pEMR515                                                                             <0.1       1.8        <0.05                                        (leu.sup.+)/a)                                                                 GRF18 pEMR515                                                                              38         5.4        23                                           (leu.sup.+)/b)                                                                 EMY500 pEMR515                                                                             20         2.5        26                                           (leu.sup.+)/b)                                                                 EMY761 pEMR515                                                                             33         4.2        26                                           (leu.sup.+)/b)                                                                 ______________________________________                                          a): the strains are cultivated in the presence of glucose (noninduction        conditions)                                                                    b): the strains are cultivated in the absence of glucose and in the            presence of galactose (induction)                                        

These results show that a high level of expression of urate oxidase can be obtained with three nonisogenic recipient strains transformed by the expression vector according to the invention.

EXAMPLE 15: Expression in a 2.5 l fermenter of the cDNA of urate oxidase for the EMY500 pEMR515 strain. Purification and partial characterization of the recombinant urate oxidase: 1) Culture in a 2.5 l fermenter of the EMY500 pEMR515 strain:

The culture of the EMY500 pEMR515 strain is carried out in the following manner:

a) Preculture stage in erlenmeyer

A 500 ml erlenmeyer containing 90 ml of a growth medium MCPA, (sterilizable by autoclave) complemented with 1.28 g of MES (2-/N-morpholino/-ethanesulfonic acid: Sigma n° M8250) and 10 ml of a growth medium MCPF (sterilized by ultra filtration) is seeded with 1 ml of a solution of the EMY500 pEMR515 strain in a medium containing 20% glycerol with a number of cells corresponding to an Optical Density of 2.35. The compositions of the media MCPA and MCPF are given hereinafter. After 24 hours of incubation, under stirring at 30° C., the Optical Density of the culture is about 7.

b) Culture phase in fermenter

The above culture is used for seeding a 2.5 l fermenter containing the culture medium having the following composition:

    900 ml of MCPA+200 ml of MCPF

The pH of the culture is regulated by the fermenter to the given value of 5,5. After 6-7 hours of culture at 30° C., 72 ml of a 500 g/l glucose solution is linearly added over a period of 9 hours (namely a total of 36 g of glucose).

c) Expression stage

To the previously described mixture, 100 ml of the expression medium MEPA (sterilizable by autoclave) and 150 ml of the expression medium MEPF (sterilized by ultra filtration) having the following compositions, are added. The culture is then continued for 5 hours. Then 150 ml of a solution containing 30 g of galactose, 15 g of glycerol and 36 g of ethanol are linearly added for 20 hours. An optical density of about 160 is then obtained.

    ______________________________________                                         CHEMICAL COMPOSITION OF THE GROWTH AND                                         EXPRESSION MEDIA -                                                             Growth medium MCPA (sterilizable by autoclave)                                                     For total 900 ml                                           ______________________________________                                         NTA (nitrilotriacetic acid)                                                                          1.2       g                                              Yeast extract (DIFCO) 6         g                                              K.sub.2 SO.sub.4      1.2       g                                              NaCl                  0.6       g                                              MgSO.sub.4, 7H.sub.2 O                                                                               1.2       g                                              CaCl.sub.2 2H.sub.2 O 840       mg                                             FeCl.sub.3            108       mg                                             glutamic acid         4.44      g                                              HYCASE SF (Sheffield Products)                                                                       30        g                                              leucine               2.16      g                                              histidine             600       mg                                             methionine            1.2       g                                              oligoelements I (see hereinafter)                                                                    5         ml                                             uracil                1.2       g                                              ______________________________________                                    

    ______________________________________                                         List of oligoelements I                                                                      for 1 l of ultra purified water                                  ______________________________________                                         CuSO.sub.4, 5H.sub.2 O                                                                         780          mg                                                H.sub.3 BO.sub.3                                                                               5            g                                                 ZnSO.sub.4, 7H.sub.2 O                                                                         3            g                                                 KI              1            g                                                 MnSO.sub.4, 2H.sub.2 O                                                                         3.5          g                                                 Na.sub.2 MO.sub.4. 2H.sub.2 O                                                                  2            g                                                 FeCl.sub.3, 6.sub.H2 O                                                                         4.8          g                                                 ______________________________________                                          Add 100 ml of concentrated hydrochloric acid to the solution and adjust t      1,000 ml.                                                                

    ______________________________________                                         Growth medium MCPF (sterilized by ultra filteration)                                            for total 200 ml of                                                            ultra purified water                                          ______________________________________                                         KH.sub.2 PO.sub.4  4.8         g                                               Tryptophane        420         mg                                              Vitamin I (see hereinafter)                                                                       5           ml                                              glucose            36          g                                               ______________________________________                                          Heat to dissolve, return to ambient temperature, add the vitamins I and        filter through 0.2 μm filter.                                         

    ______________________________________                                         List of vitamins I                                                                             for total 100 ml of                                                            ultra purified water                                           ______________________________________                                         biotine           1.2         mg                                               folic acid        1           mg                                               niacine           144         mg                                               (nicotinic acid)                                                               pyridoxine.HCl    60          mg                                               thiamine.HCl      240         mg                                               calcium pantothenate                                                                             1.2         g                                                mesoinositol      2.4         g                                                ______________________________________                                          Fill to 100 ml after dissolving                                                Sterile filter, cold, at 0.2 μm                                       

    ______________________________________                                         Expression medium MEPA (sterilizable by autoclave)                                                 for total 100 ml of                                                            ultra purified water                                       ______________________________________                                         NTA                   1.2       g                                              K2 so4                2.08      g                                              glutamic acid         6         g                                              HYCASE SF (Sheffield Products)                                                                       24        g                                              leucine               2.16      g                                              histidine             600       mg                                             methionine            1.2       g                                              MgSO.sub.4, 7H.sub.2 O                                                                               720       mg                                             CaCl.sub.2, 2H.sub.2 O                                                                               840       mg                                             FeCl.sub.3, 6H.sub.2 O                                                                               108       mg                                             oligoelements I       5         ml                                             uracil                1.2       g                                              ______________________________________                                          Adjust the pH to 5.5 with concentrated H.sub.2 SO.sub.4 or concentrated        KOH                                                                            Autoclave for 20 mins at 120° C.                                  

    ______________________________________                                         Expression medium MEPF (sterilized by ultra filtration)                                    for total 150 ml of ultra purified water                           ______________________________________                                         KH.sub.2 PO.sub.4                                                                            2.4           g                                                  tryptophane   420           mg                                                 vitamins I    5             ml                                                 glycerol      36            g                                                  galactose     45            g                                                  ______________________________________                                          Heat to dissolve, return to ambient temperature, add the vitamins and          filter.                                                                  

2) Grinding of the cells

After 20 hours of induction, the OD of the culture, measured at 600 nm, is 98. 800 g of the fermentation wort are centrifugated for 5 minutes at 10,000 g and the cell cake is taken up in 80 ml of a lysis buffer (glycine 20 mM pH 8.5). The cells are then ground twice at 4° C., for 2.5 minutes in a grinding device (Vibrogenic Zellmuhle mill V14) in the presence of a volume of beads (0.50 mm in diameter) equal to that of the solution of cells to be lysed. After grinding, the supernatant is taken up and the beads are washed twice with 80 ml of a lysis buffer. 210 ml of a lysate are recovered; said lysate has a total protein content of about 3 mg/ml and a urate oxydase activity of about 7.7 U/ml (namely a urate oxidase percentage towards the total protein of about 8.5%, considering a specific activity of that protein of 30 U/mg).

3) Purification of the recombinant urate oxidase

a) Purification protocol

The above lysate is submitted to the two-step purification protocol disclosed hereinafter.

Step 1: Anionic chromatography

Support

DEAE (diethylaminosulphate) sepharose fast flow (Pharmacia ref. 17.07.09.91)

The compressed gel occupies a volume of 70 ml.

The separation is carried out at ambient temperature, the recovered fractions being preserved at 0° C.

Separation conditions

A gradient of a chloride ionic force between buffer 1 (sodium borate 10 mM, pH 9.2) and buffer 2 (sodium borate 10 mM, sodium chloride 1M) is used. The buffers are previously degased and preserved at O° C. during the elution. In each buffer 0.02% of azide are added.

The raw extract is deposited (10 ml) and eluted with buffer 1 up to the complete recovery of the urate oxidase (by fractions of 10 ml) which is not retained on the column.

The pigments and the contaminating proteins are thereafter removed by an elution with buffer 2.

The purification is followed by measuring of the OD of the eluate at 214 nm.

Step 2: High pressure and inverse phase liquid chromatography

Support

Grafted C8 silica column, Aquapore OD-300 (100×2.1 mm) (Brownlee-Applied Biosystems)

Operating conditions

Eluent 1: ultrapurified water (filtered through a Millipore system) containing 0.1% of trifluoroacetic acid.

Eluent 2: Acetonitrile (of spectrophotometric quality or similar) containing 0.08% of trifluoroacetic acid.

Flow rate: 0.3 ml/min.

The gradient is of 35% of acetonitrile/TFA to 70% of acetonitrile/TFA for 20 minutes and is maintained at 70% for 5 minutes. The injected quantity is of 1 ml per run.

Recovery of the fractions

The separation is followed by measurement of the optical density at 218 nm. The acetonitrile is evaporated during the centrifugation under vacuum.

b) Results

The sample before and after the first step of purification was analysed by liquid chromatography on a grafted C8 silica column, the Aquapore OD-300 previously disclosed with the same gradient, with an injected quantity of 50 μl . Purified urate oxidase from A flavus is used as an external control.

In the starting lysate, the urate oxidase represents 63% of the total proteins. After the first step of purification, the urate oxidase represents 84% of the total proteins.

The whole sample obtained after step 2 was used for the following partial characterization. Said sample certainly contains more than 84% of urate oxidase.

4) Partial characterization of the recombinant urate oxidase

a) Analysis of the amino acids

The analysis of the amino acids of the acid hydrolysate of the purified recombinant urate oxidase was carried out in an analyser from Applied Biosystems model 420-130A. The distribution of the quantified amino acids is compatible (there exists no significant difference) with the supposed sequence. The same result was observed for the purified urate oxidase extracted from A. flavus (obtained in example 4)

b) Tryptic peptidic map

A tryptic peptidic map was established for the purified recombinant urate oxidase and for the purified urate oxidase extract obtained in example 4) under the following conditions:

A urate oxidase solution having a concentration of 1 mg/ml is prepared. Extemporaneously a trypsin solution having a concentration of 1 mg/ml is prepared.

The two solutions are mixed together in a proportion of 1/30 enzyme/substrate for 8 hours at ambient temperature. The tryptic hydrolysate is then chromatographied (liquid phase chromatography) on a C18 grafted silica column (5 μm; lichrosorb 250×4.6 mm Hichrom-ref. RP 18-5-250A) provided with a UV detector coupled with a recorder. The gradient applied is of 1% acetonitrile/TFA to 60% acetonitrile/TFA for 120 minutes and then the gradient is maintained at 60% for 5 minutes.

The peptidic maps obtained have a very narrow profile.

5) Determination of the blocked character of the amino-terminal sequence

The amino-terminal sequence was analysed by means of the sequencer, Applied Biosystem model 470A, coupled with an analyser of phenylthiohydantoic derivatives, Applied Biosystems model 120A. The purified recombinant urate oxidase (200 pmoles detected by analysis of the amino acids) was put on the sequencer in the presence of 20 pmoles of β-lactoglobuline (control protein).

No amino-terminal sequence corresponding to the sequence of the urate oxidase was detected, whereas the amino-terminal sequence of the control protein was detected.

Therefore, the recombinant urate oxidase of the invention, as well as the urate oxidase extract, has a blocked amino-terminal end.

EXAMPLE 16: Construction of an expression vector for urate oxidase cDNA in animal cells: plasmid pSV860

This vector was obtained by

ligation of the small AccI-SnaBI fragment containing a sequence coding for urate oxidase with the exception of the first 16 amino acids, said fragment being derived from plasmid p466 (an expression vector for A. flavus urate oxidase in E. coli, available in the laboratory and described below), with a synthetic HindIII-AccI fragment, which made it possible to obtain a HindIII-SnaBI fragment containing a complete sequence coding for A. flavus urate oxidase and a non-translated 5' sequence favoring expression in animal cells; and

insertion of the HindIII-SnaBI fragment between the HindIII and SnaBI sites of the multiple cloning site (also called polylinker) of the expression vector for animal cells, namely plasmid pSE₁.

The following account will successively describe the construction of plasmid p466, plasmid pSE₁ and plasmid pSV860.

1) Construction of plasmid p466

Plasmid p466, an expression vector for urate oxidase cDNA in E. coli, was prepared. It comprises a fragment of pBR327 including the origin of replication and the ampicillin resistance gene; it also comprises a synthetic promoter of E. coli (R. RODRIGUEZ and M. CHAMBERLIN, "Promoters-Structure and function (1982), Preager), a Shine-Dalgarno sequence followed by a polylinker containing the unique NdeI and KpnI sites, a transcription terminator (derived from phage fd) and the lac i gene.

This plasmid was constructed from an expression plasmid for hGH in E. coli (p462) by replacing a fragment carrying the hGH gene with urate oxidase cDNA.

The construction of plasmid p466 was described in detail in Example 7 above.

2) Construction of an expression vector for animal cells: plasmid pSE₁

The strategy employed uses fragments obtained from pre-existing plasmids available to the public, and fragments prepared synthetically by the techniques now in common use. The cloning techniques employed are those described by T. MANIATIS, E. F. FRITSCH and J. SAMBROOK in "Molecular Cloning, a laboratory manual" (Cold Spring Harbor Laboratory, 1984). The oligonucleotides are synthesized with the aid of a Biosearch 4600 DNA synthesizer.

The following description will be understood more clearly with reference to FIG. 13, which shows a restriction map of plasmid pSE₁, the sites which have disappeared due to ligation being indicated in brackets. The symbols used in this Figure will be specified in the description below.

This plasmid was constructed by successive ligations of the following components:

1)--a PvuII-PvuII fragment--symbolized by in FIG. 13--of 2525 bp, obtained by complete digestion of plasmid pTZ18R (Pharmacia) with the restriction enzyme PvuII. This fragment contains the origin of replication of phage F1 (denoted by ORI F1 in FIG. 13), a gene (denoted by Amp^(R) in FIG. 13) carrying ampicillin resistance, and the origin of replication (denoted by ORI pBR322 in FIG. 13) permitting the replication of this plasmid in E. coli. The first PvuII blunt site disappears on ligation with the EcoRV blunt site (which also disappears) of the fragment described in 7).

2)--a PvuII-HpaI fragment--symbolized by in FIG. 13--of 1060 bp, of type 5 adenovirus DNA between position 11299 (PvuII restriction site) and position 10239 (HpaI restriction site) (DEKKER & VAN ORMONDT, Gene 27, 1984, 115-120), containing the information for VA-I and VA-II RNA's. The HpaI blunt site disappears on ligation with the PvuII blunt site (which also disappears) of the fragment described in 3).

3)--a PvuII-HindIII fragment--symbolized by in FIG. 13--of 344 bp, derived from SV40 virus DNA and obtained by complete digestion with the restriction enzymes PvuII and HindIII. This fragment contains the origin of replication and the early promoter of SV40 virus DNA (ref. B. J. BYRNE et al., PNAS-USA (1983) 80, 721-725).

The HindIII site disappears on ligation with the site binding to HindIII of the fragment described in 4).

4)--a synthetic "site binding to HindIII"-HindIII fragment--symbolized by in FIG. 13--of 419 bp, whose sequence, given below (SEQUENCE ID. NO. 33), is similar to the nontranslated 5' sequence of the HTLV1 virus (ref. WEISS et al., "Molecular Biology of Tumor Viruses"-part 2-2nd edition-1985-Cold Spring Harbor Laboratory-p. 1057). ##STR16## 5)--a synthetic HindIII-"site binding to BamHI" fragment--symbolized by in FIG. 13--containing the promoter of the RNA polymerase of phage T7 and also a polylinker containing the SmaI cloning site and having the sequence below (SEQUENCE ID NO. 34). ##STR17## 6)--a BamHI-BcII fragment of 240 bp--represented by in FIG. 13--which is a small fragment obtained by complete digestion of the SV40 virus with the enzymes BcII and BamHI and containing the late polyadenylation site of said virus (M. FITZGERALD et al., Cell, 24, 1981, 251-260). The BamHI and BcII sites disappear on ligation respectively with the site binding to BamHI of the fragment described in 5) and the BamHI site (which also disappears) of the fragment described in 7).

7)--a BamHI-EcoRV fragment--symbolized by in FIG. 13--of 190 bp, which is a small fragment derived from plasmid pBR322 after complete digestion with the enzymes EcoRV and BamHI.

3) Construction of plasmid pSV860

Plasmid p466 (cf. FIG. 9) was completely digested with the enzymes AccI and SnaBI. The small AccI-SnaBI fragment, which contains a DNA sequence coding for urate oxidase with the exception of the first 16 amino-terminal acids, was purified and ligated with the synthetic HindIII-AccI fragment having the following sequence (SEQUENCE ID NO. 35): ##STR18##

This ligation makes it possible to obtain the HindIII-SnaBI fragment containing a sequence, coding for urate oxidase, which is identical to that of clone 9C and a non-translated 5' sequence favoring expression in animal cells (KOZAK, M., Nucl. Acids Res., 12, 2, 1984, 857-872).

The HindIII-SnaBI fragment contains the following sequence (SEQUENCE ID NO. 36):

    __________________________________________________________________________     5'                                                                             AGCTTGCCG   CCACTATGTC                                                                               CGCAGTAAAA                                                                               GCAGCCCGCT                                                                               ACGGCAAGGA                             CAATGTCCGC                                                                               GTCTACAAGG                                                                               TTCACAAGGA                                                                               CGAGAAGACC                                                                               GGTGTCCAGA                             CGGTGTACGA                                                                               GATGACCGTC                                                                               TGTGTGCTTC                                                                               TGGAGGGTGA                                                                               GATTGAGACC                             TCTTACACCA                                                                               AGGCCGACAA                                                                               CAGCGTCATT                                                                               GTCGCAACCG                                                                               ACTCCATTAA                             GAACACCATT                                                                               TACATCACCG                                                                               CCAAGCAGAA                                                                               CCCCGTTACT                                                                               CCTCCCGAGC                             TGTTCGGCTC                                                                               CATCCTGGGC                                                                               ACACACTTCA                                                                               TTGAGAAGTA                                                                               CAACCACATC                             CATGCCGCTC                                                                               ACGTCAACAT                                                                               TGTCTGCCAC                                                                               CGCTGGACCC                                                                               GGATGGACAT                             TGACGGCAAG                                                                               CCACACCCTC                                                                               ACTCCTTCAT                                                                               CCGCGACAGC                                                                               GAGGAGAAGC                             GGAATGTGCA                                                                               GGTGGACGTG                                                                               GTCGAGGGCA                                                                               AGGGCATCGA                                                                               TATCAAGTCG                             TCTCTGTCCG                                                                               GCCTGACCGT                                                                               GCTGAAGAGC                                                                               ACCAACTCGC                                                                               AGTTCTGGGG                             CTTCCTGCGT                                                                               GACGAGTACA                                                                               CCACACTTAA                                                                               GGAGACCTGG                                                                               GACCGTATCC                             TGAGCACCGA                                                                               CGTCGATGCC                                                                               ACTTGGCAGT                                                                               GGAAGAATTT                                                                               CAGTGGACTC                             CAGGAGGTCC                                                                               GCTCGCACGT                                                                               GCCTAAGTTC                                                                               GATGCTACCT                                                                               GGGCCACTGC                             TCGCGAGGTC                                                                               ACTCTGAAGA                                                                               CTTTTGCTGA                                                                               AGATAACAGT                                                                               GCCAGCGTGC                             AGGCCACTAT                                                                               GTACAAGATG                                                                               GCAGAGCAAA                                                                               TCCTGGCGCG                                                                               CCAGCAGCTG                             ATCGAGACTG                                                                               TCGAGTACTC                                                                               GTTGCCTAAC                                                                               AAGCACTATT                                                                               TCGAAATCGA                             CCTGAGCTGG                                                                               CACAAGGGCC                                                                               TCCAAAACAC                                                                               CGGCAAGAAC                                                                               GCCGAGGTCT                             TCGCTCCTCA                                                                               GTCGGACCCC                                                                               AACGGTCTGA                                                                               TCAAGTGTAC                                                                               CGTCGGCCGG                             TCCTCTCTGA                                                                               AGTCTAAATT                                                                               G                                                        __________________________________________________________________________

The HindIII-SnaBI fragment was then inserted into vector pSE₁, which had first been incubated with the enzymes HindIII and SmaI. This gave plasmid pSV860 shown in FIG. 14, in which the symbols have the same meanings as in FIG. 13, the novel HindIII-SnaBI fragment being symbolized by . (The SnaBI and SmaI sites disappeared on ligation.)

EXAMPLE 17: Transient expression of urate oxidase cDNA in COS cells-Assay of the urate oxidase activity in the cell lyzate

COS cells are monkey kidney cells expressing the T-antigen of the SV40 virus (Gluzman, Y., Cell 23, 1981, 175-182). These cells, which permit the replication of vectors containing the origin of replication of SV40 virus DNA, are preferred hosts for studying the expression of genes in animal cells.

1) Transfection of COS cells and transient expression of urate oxidase cDNA

4.10⁵ COS cells are plated out in a Petri dish of diameter 6 cm (Corning) in 5 ml of Dulbecco's modified Eagle's medium (from Gibco), hereafter called DMEM, which contains 0.6 g/l of glutamine and 3.7 g/l of NaHCO₃ and is complemented with fetal calf serum (GIBCO) at a rate of 5%. After about 16 h of culture at 37° C. in an atmosphere containing 5% of carbon dioxide, the culture medium is sucked off and the cells are washed with 3 ml of PBS (phosphate buffered saline from GIBCO). The following mixture is then added: 1000 μl of (DMEM+10% of fetal calf serum (GIBCO)), 110 μl of diethylaminoethyldextran of average molecular weight 500,000 at a concentration of 2 mg/ml (Pharmacia), 1.1 μl of chloroquine 100 mM (Sigma) and 3 μg of DNA of either plasmid pSV860 or plasmid pSE₁ (for the control). After incubation for 5 h at 37° C. in an atmosphere containing 5% of carbon dioxide, the mixture is withdrawn from the cells. 2 ml of PBS containing 10% of dimethyl sulfoxide (spectroscopic grade, Merck) are then added. After incubation for 1 min at room temperature, the mixture is withdrawn and the cells are washed twice with PBS. 5 ml of DMEM complemented with fetal calf serum at a rate of 2% are added. Incubation is continued for 4 days at 37° C. under an atmosphere containing 5% of carbon dioxide.

2) Preparation of the samples

The culture medium is sucked off and the COS cells are rinsed twice with 3 ml of PBS. The cells are then collected by scratching with a rubber spatula (policeman) in 1 ml of PBS. After scratching, the dish is rinsed with 1 ml of PBS. The two cell suspensions are combined and centrifuged for 10 min at 1000 rpm. The supernatant is removed and the cell residue is resuspended in 1 ml of triethylammonium (TEA) 0.05M of pH 8.9/EDTA buffer.

The cells are lyzed by sonication (on ice) by means of 10 s pulses with a sonicator (Vibra Cell from Sonics and Materials Inc. USA) set to a power of 12 W. The cell lyzate is centrifuged for 10 min at 10,000 rpm and the supernatant is recovered for assay of the urate oxidase.

3) Assay of the urate oxidase activity

The urate oxidase activity was assayed as described in Example 9.

The results are collated in the Table below:

    ______________________________________                                         COS cells transfected                                                                          Urate oxidase activity                                         by              U/ml                                                           ______________________________________                                         pSVS60          0.105                                                          pSE.sub.1       <0.01                                                          ______________________________________                                    

It is found that the COS cells transfected by plasmid pSV860 carrying urate oxidase cDNA express an appreciable level of urate oxidase activity, whereas no urate oxidase activity is detectable in the control. There is therefore expression of urate oxidase cDNA.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 36                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 301 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Aspergillus flavus                                               (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Urate oxidase                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        SerAlaValLysAlaAlaArgTyrGlyLysAspAsnValArgValTyr                               151015                                                                         LysValH isLysAspGluLysThrGlyValGlnThrValTyrGluMet                              202530                                                                         ThrValCysValLeuLeuGluGlyGluIleGluThrSerTyrThrLys                                354045                                                                        AlaAspAsnSerValIleValAlaThrAspSerIleLysAsnThrIle                               505560                                                                         TyrIleThrAlaLysGlnA snProValThrProProGluLeuPheGly                              65707580                                                                       SerIleLeuGlyThrHisPheIleGluLysTyrAsnHisIleHisAla                                859095                                                                        AlaHisValAsnIleValCysHisArgTrpThrArgMetAspIleAsp                               100105110                                                                      GlyLysProHis ProHisSerPheIleArgAspSerGluGluLysArg                              115120125                                                                      AsnValGlnValAspValValGluGlyLysGlyIleAspIleLysSer                               130 135140                                                                     SerLeuSerGlyLeuThrValLeuLysSerThrAsnSerGlnPheTrp                               145150155160                                                                   GlyPheLeuArgAspG luTyrThrThrLeuLysGluThrTrpAspArg                              165170175                                                                      IleLeuSerThrAspValAspAlaThrTrpGlnTrpLysAsnPheSer                               18 0185190                                                                     GlyLeuGlnGluValArgSerHisValProLysPheAspAlaThrTrp                               195200205                                                                      AlaThrAlaArgGluVa lThrLeuLysThrPheAlaGluAspAsnSer                              210215220                                                                      AlaSerValGlnAlaThrMetTyrLysMetAlaGluGlnIleLeuAla                               225230 235240                                                                  ArgGlnGlnLeuIleGluThrValGluTyrSerLeuProAsnLysHis                               245250255                                                                      TyrPheGluIleAsp LeuSerTrpHisLysGlyLeuGlnAsnThrGly                              260265270                                                                      LysAsnAlaGluValPheAlaProGlnSerAspProAsnGlyLeuIle                               275 280285                                                                     LysCysThrValGlyArgSerSerLeuLysSerLysLeu                                        290295300                                                                      (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 302 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Aspergillus flavus                                               (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Met-Urate oxidase                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetSerAlaValLysAlaAlaArgTyrGlyLys AspAsnValArgVal                              151015                                                                         TyrLysValHisLysAspGluLysThrGlyValGlnThrValTyrGlu                               2025 30                                                                        MetThrValCysValLeuLeuGluGlyGluIleGluThrSerTyrThr                               354045                                                                         LysAlaAspAsnSerValIleValAlaThrAspSer IleLysAsnThr                              505560                                                                         IleTyrIleThrAlaLysGlnAsnProValThrProProGluLeuPhe                               657075 80                                                                      GlySerIleLeuGlyThrHisPheIleGluLysTyrAsnHisIleHis                               859095                                                                         AlaAlaHisValAsnIleValCysHisArgTrpThr ArgMetAspIle                              100105110                                                                      AspGlyLysProHisProHisSerPheIleArgAspSerGluGluLys                               115120 125                                                                     ArgAsnValGlnValAspValValGluGlyLysGlyIleAspIleLys                               130135140                                                                      SerSerLeuSerGlyLeuThrValLeuLysSerThrAsnSerGln Phe                              145150155160                                                                   TrpGlyPheLeuArgAspGluTyrThrThrLeuLysGluThrTrpAsp                               165170 175                                                                     ArgIleLeuSerThrAspValAspAlaThrTrpGlnTrpLysAsnPhe                               180185190                                                                      SerGlyLeuGlnGluValArgSerHisValProLysP heAspAlaThr                              195200205                                                                      TrpAlaThrAlaArgGluValThrLeuLysThrPheAlaGluAspAsn                               210215220                                                                       SerAlaSerValGlnAlaThrMetTyrLysMetAlaGluGlnIleLeu                              225230235240                                                                   AlaArgGlnGlnLeuIleGluThrValGluTyrSerLeuPr oAsnLys                              245250255                                                                      HisTyrPheGluIleAspLeuSerTrpHisLysGlyLeuGlnAsnThr                               260265 270                                                                     GlyLysAsnAlaGluValPheAlaProGlnSerAspProAsnGlyLeu                               275280285                                                                      IleLysCysThrValGlyArgSerSerLeuLysSerLysLeu                                     290295300                                                                      (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 906 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Preferred sequence for expression in                                 prokaryotes                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATGTCTGCGGTAAAAGCAGCGCGCTACGGCAAGGACAATGTTCGCGTCTACAAGGTTCAC60                 AAGGACGAGAAGACCGGTGTCCAGACGGTGTACGAGATGACCGTCTGTGTGCTTCTGGAG120                GGTGAGATTGAGA CCTCTTACACCAAGGCCGACAACAGCGTCATTGTCGCAACCGACTCC180               ATTAAGAACACCATTTACATCACCGCCAAGCAGAACCCCGTTACTCCTCCCGAGCTGTTC240                GGCTCCATCCTGGGCACACACTTCATTGAGAAGTACAACCACATCCATGCCGCTCACGT C300               AACATTGTCTGCCACCGCTGGACCCGGATGGACATTGACGGCAAGCCACACCCTCACTCC360                TTCATCCGCGACAGCGAGGAGAAGCGGAATGTGCAGGTGGACGTGGTCGAGGGCAAGGGC420                ATCGATATCAAGTCGTCTCTGTCCGGCCTGACCGTG CTGAAGAGCACCAACTCGCAGTTC480               TGGGGCTTCCTGCGTGACGAGTACACCACACTTAAGGAGACCTGGGACCGTATCCTGAGC540                ACCGACGTCGATGCCACTTGGCAGTGGAAGAATTTCAGTGGACTCCAGGAGGTCCGCTCG600                CACGTGCCTAAGT TCGATGCTACCTGGGCCACTGCTCGCGAGGTCACTCTGAAGACTTTT660               GCTGAAGATAACAGTGCCAGCGTGCAGGCCACTATGTACAAGATGGCAGAGCAAATCCTG720                GCGCGCCAGCAGCTGATCGAGACTGTCGAGTACTCGTTGCCTAACAAGCACTATTTCGA A780               ATCGACCTGAGCTGGCACAAGGGCCTCCAAAACACCGGCAAGAACGCCGAGGTCTTCGCT840                CCTCAGTCGGACCCCAACGGTCTGATCAAGTGTACCGTCGGCCGGTCCTCTCTGAAGTCT900                AAATTG 906                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 906 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Preferred sequence for expression in                                eukaryotes                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGTCTGCTGTTAAGGCTGCTAGATACGGTAAGGACAACGTTAGAGTCTACAAGGTTCAC60                 AAGGACGAGAAGACCGGTGTCCAGACGGTGTACGAGATGACCGTCTGTGTGCTTCTGGAG120                GGTGAGATTGAGACCTCTTACACCAAGGCCGA CAACAGCGTCATTGTCGCAACCGACTCC180               ATTAAGAACACCATTTACATCACCGCCAAGCAGAACCCCGTTACTCCTCCCGAGCTGTTC240                GGCTCCATCCTGGGCACACACTTCATTGAGAAGTACAACCACATCCATGCCGCTCACGTC300                AACATTGTCT GCCACCGCTGGACCCGGATGGACATTGACGGCAAGCCACACCCTCACTCC360               TTCATCCGCGACAGCGAGGAGAAGCGGAATGTGCAGGTGGACGTGGTCGAGGGCAAGGGC420                ATCGATATCAAGTCGTCTCTGTCCGGCCTGACCGTGCTGAAGAGCACCAACTCGC AGTTC480               TGGGGCTTCCTGCGTGACGAGTACACCACACTTAAGGAGACCTGGGACCGTATCCTGAGC540                ACCGACGTCGATGCCACTTGGCAGTGGAAGAATTTCAGTGGACTCCAGGAGGTCCGCTCG600                CACGTGCCTAAGTTCGATGCTACCTGGGCCAC TGCTCGCGAGGTCACTCTGAAGACTTTT660               GCTGAAGATAACAGTGCCAGCGTGCAGGCCACTATGTACAAGATGGCAGAGCAAATCCTG720                GCGCGCCAGCAGCTGATCGAGACTGTCGAGTACTCGTTGCCTAACAAGCACTATTTCGAA780                ATCGACCTGA GCTGGCACAAGGGCCTCCAAAACACCGGCAAGAACGCCGAGGTCTTCGCT840               CCTCAGTCGGACCCCAACGGTCTGATCAAGTGTACCGTCGGCCGGTCCTCTCTGAAGTCT900                AAATTG 906                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Preferred non-translated 5'sequence for                             animal cells                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        AGCTTGCCGCCACT14                                                               (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 906 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ii i) HYPOTHETICAL: NO                                                        (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Preferred sequence for expression in animal                         cells                                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        ATGTCCGCAGTAAAAGCAGCCCGCTACGGCAAGGACAATGTCCGCGTCTACAAGGTTCAC60                 AAGGACGAGAAGACCGGTGTCCAGACGGTGTACGAGATGACCGTC TGTGTGCTTCTGGAG120               GGTGAGATTGAGACCTCTTACACCAAGGCCGACAACAGCGTCATTGTCGCAACCGACTCC180                ATTAAGAACACCATTTACATCACCGCCAAGCAGAACCCCGTTACTCCTCCCGAGCTGTTC240                GGCTCCATCCTGGGCACACACT TCATTGAGAAGTACAACCACATCCATGCCGCTCACGTC300               AACATTGTCTGCCACCGCTGGACCCGGATGGACATTGACGGCAAGCCACACCCTCACTCC360                TTCATCCGCGACAGCGAGGAGAAGCGGAATGTGCAGGTGGACGTGGTCGAGGGCAAGGGC420                 ATCGATATCAAGTCGTCTCTGTCCGGCCTGACCGTGCTGAAGAGCACCAACTCGCAGTTC480               TGGGGCTTCCTGCGTGACGAGTACACCACACTTAAGGAGACCTGGGACCGTATCCTGAGC540                ACCGACGTCGATGCCACTTGGCAGTGGAAGAATTTCAGTGGACTC CAGGAGGTCCGCTCG600               CACGTGCCTAAGTTCGATGCTACCTGGGCCACTGCTCGCGAGGTCACTCTGAAGACTTTT660                GCTGAAGATAACAGTGCCAGCGTGCAGGCCACTATGTACAAGATGGCAGAGCAAATCCTG720                GCGCGCCAGCAGCTGATCGAGA CTGTCGAGTACTCGTTGCCTAACAAGCACTATTTCGAA780               ATCGACCTGAGCTGGCACAAGGGCCTCCAAAACACCGGCAAGAACGCCGAGGTCTTCGCT840                CCTCAGTCGGACCCCAACGGTCTGATCAAGTGTACCGTCGGCCGGTCCTCTCTGAAGTCT900                 AAATTG906                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: reverse transcription primer                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        GATCCGGGCCCTTTTTTTTTTTT23                                                      (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 17                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        AsnValGlnValAspValValGluGlyLys                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                                (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 20                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        AsnPheSerGlyLeuGlnGluVal                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 6 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 23                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       P heAspAlaThrTrpAla                                                            15                                                                             (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 27                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       HisTyrPheGluIleAspLeuSer                                                       15                                                                             (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 13 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ii i) HYPOTHETICAL: NO                                                        (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 28                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       IleLeuSerThrAspValAspAlaThrTrpGlnTrpLys                                        1510                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 29                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       HisTyrPheGluIleAspLeuSerTrpHisLys                                              1 510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 31                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                        SerThrAsnSerGlnPheTrpGlyPheLeuArg                                             1510                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 32                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       GlnAsnProValThrProProGluLeuPheGlySerIleLeuGlyThr                               15101 5                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product T 33                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       GlnAsnProValThr ProProGluLeuPheGlySerIleLeuGlyThr                              151015                                                                         (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product V 1                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       TyrSerLeuProAsnLysHisTyrPheGluIleAspLeuSerTrpHis                               1510 15                                                                        Lys                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product V 2                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       ValThrLeuLysThrPheAlaGluAspAsnSerAlaSerValGlnAla                               151015                                                                         (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 amino acids                                                     (B) TYPE: amino acid                                                            (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product V 3                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       ThrSerTyrThrLysAlaAspAsnSerValIleValAspThrAspSer                               1 51015                                                                        IleLysAsnThrIleTyrIleThr                                                       20                                                                             (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydrolysis product V 5                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       GlyLysGlyIleAspIleLysSerSerLeuSerGlyLeuThrValLeu                               1510 15                                                                        LysSerThrAsnSerGlnPheTrpGlyPheLeuArg                                           2025                                                                           (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       ( D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Hydolysis product V 6                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       GlyLysGlyIleAspIleLysSerSerLeuSerGlyLeuThrValLeu                               1510 15                                                                        Lys                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1236 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Fragment 3                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                        GATCCGCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTTACATT60                AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA120                ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG CCAGGGTGGTTTTTC180               TTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTT240                GCAGCAAGCGGTCCACGCTGGTTTGCCCCACCACCCGAAAATCCTGTTTGATGGTGGTTA300                ACGGCGGGATATAACATGAGCT GTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCG360               CACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGT420                TGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAA480                 AACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAG540               TGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCG600                CTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCA CGCCCAGTCGCGTAC660               CGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATA720                ACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGAT780                AGTTAATGATCAGCCCACTGAC GCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGG840               CTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGC900                GAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAA960                 CGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAAT1020              TCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCT1080               GGTTCACCACGCGGGAAACGGTCTGATAACAGACACCGGCATACT CTGCGACATCGTATA1140              ACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCA1200               TACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCG1236                                       (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                   (A) LENGTH: 321 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Fragment 4                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 107..316                                                         (D) OTHER INFORMATION: /product="regulatory signal aa                           1-44 human growth hormone precursor"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       TCGAGCTGACTGACCTGTTGCTTATATTACATCGATAGCGTATAATGTGTGGAATTGTGA60                 GCGATAACAATTTCACACAGTTTAACTTTAAGAAGGAGATATACATATGGCTACC115                      MetAlaThr                                                                     GGATCCCGGACTAGTCTGCTCCTGGCTTTTGGCCTGCTCTGCCTGCCC163                            GlySerArgThr SerLeuLeuLeuAlaPheGlyLeuLeuCysLeuPro                              51015                                                                          TGGCTTCAAGAGGGCAGTGCCTTCCCAACCATTCCCTTATCTAGACTT211                            TrpLeuGlnGluGlySerAla PheProThrIleProLeuSerArgLeu                              20253035                                                                       TTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTT259                            PheAspAsnAlaMetLe uArgAlaHisArgLeuHisGlnLeuAlaPhe                              404550                                                                         GACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAG307                            AspThrTyrGlnGluP heGluGluAlaTyrIleProLysGluGlnLys                              556065                                                                         TATTCATTCCTGCA321                                                              TyrSerPhe                                                                      70                                                                             (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 70 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       MetAlaThrGlySerArgThrSerLeuLeuLeuAlaPheGlyLeuLeu                               15 1015                                                                        CysLeuProTrpLeuGlnGluGlySerAlaPheProThrIleProLeu                               202530                                                                         SerArgLeuPheAspAsnAlaMetLeuAr gAlaHisArgLeuHisGln                              354045                                                                         LeuAlaPheAspThrTyrGlnGluPheGluGluAlaTyrIleProLys                               505560                                                                         Glu GlnLysTyrSerPhe                                                            6570                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 74 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: ClaI-NdeI fragment                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       CGATAGCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGTTTTTCGCGAA60                 GAAGGAGATATACA74                                                               (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                   (A) LENGTH: 190 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Plasmid p373,2 fragment                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       GATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCA CTACT60                CAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCT120                GCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGTAAGGTACCCTGCC180                CTACGTACCA 190                                                                 (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 48 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: AccI-NdeI synthetic fragment                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TATGTCTGCGGTAAAAGCAGCGCGCTACGGCAAGGACAATGTTCGCGT48                             (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 360 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                               (iii) HYPOTHETICAL: NO                                                        (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Plasmid pEMR469 fragment                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       GGGACGCGTCTCCTCTGCCGGAACACCGGGCATCTCCAACTTATAAGTTGGAGAAATAAG60                 AGAATTTCAGATTGAGAGAATGAAAAAAAAAAAAAAAAAAAAGGCAGAGGAGAGCATAGA 120               AATGGGGTTCACTTTTTGGTAAAGCTATAGCATGCCTATCACATATAAATAGAGTGCCAG180                TAGCGACTTTTTTCACACTCGAGATACTCTTACTACTGCTCTCTTGTTGTTTTTATCACT240                TCTTGTTTCTTCTTGGTAAATAGAATATCAAGCTACAA AAAGCATACAATCAACTATCAA300               CTATTAACTATATCGATACCATATGGATCCGTCGACTCTAGAGGATCGTCGACTCTAGAG360                (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Fragment C                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       CGATATACACAATGTCTGCTGTTAAGGCTGCTAGATACGGTAAGGACAACGTTAGAGT58                   (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1013 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Fragment D                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       CTACAAGGTTCACAAGGACGAGAAGACCGGTGTCCAGACGGTGTACGAGATGACCGTCTG60                  TGTGCTTCTGGAGGGTGAGATTGAGACCTCTTACACCAAGGCCGACAACAGCGTCATTGT120               CGCAACCGACTCCATTAAGAACACCATTTACATCACCGCCAAGCAGAACCCCGTTACTCC180                TCCCGAGCTGTTCGGCTCCATCCTGGGCACACACTTCATTGAGAA GTACAACCACATCCA240               TGCCGCTCACGTCAACATTGTCTGCCACCGCTGGACCCGGATGGACATTGACGGCAAGCC300                ACACCCTCACTCCTTCATCCGCGACAGCGAGGAGAAGCGGAATGTGCAGGTGGACGTGGT360                CGAGGGCAAGGGCATCGATATC AAGTCGTCTCTGTCCGGCCTGACCGTGCTGAAGAGCAC420               CAACTCGCAGTTCTGGGGCTTCCTGCGTGACGAGTACACCACACTTAAGGAGACCTGGGA480                CCGTATCCTGAGCACCGACGTCGATGCCACTTGGCAGTGGAAGAATTTCAGTGGACTCCA540                 GGAGGTCCGCTCGCACGTGCCTAAGTTCGATGCTACCTGGGCCACTGCTCGCGAGGTCAC600               TCTGAAGACTTTTGCTGAAGATAACAGTGCCAGCGTGCAGGCCACTATGTACAAGATGGC660                AGAGCAAATCCTGGCGCGCCAGCAGCTGATCGAGACTGTCGAGTA CTCGTTGCCTAACAA720               GCACTATTTCGAAATCGACCTGAGCTGGCACAAGGGCCTCCAAAACACCGGCAAGAACGC780                CGAGGTCTTCGCTCCTCAGTCGGACCCCAACGGTCTGATCAAGTGTACCGTCGGCCGGTC840                CTCTCTGAAGTCTAAATTGTAA ACCAACATGATTCTCACGTTCCGGAGTTTCCAAGGCAA900               ACTGTATATAGTCTGGGATAGGGTATAGCATTCATTCACTTGTTTTTTACTTCCAAAAAA960                AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGCCCG1013                      (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 207 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Synthetic GAL7 fragment                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       CGCGTCTATACTTCGGAGCACTGTT GAGCGAAGGCTCATTAGATATATTTTCTGTCATTT60                TCCTTAACCCAAAAATAAGGGAGAGGGTCCAAAAAGCGCTCGGACAACTGTTGACCGTGA120                TCCGAAGGACTGGCTATACAGTGTTCACAAAATAGCCAAGCTGAAAATAATGTGTAGCCT180                TT AGCTATGTTCAGTTAGTTTGGCATG207                                                (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Modified XbaI-MluI adapter                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       CTAGGCTAGCGGGCCCGCATGCA23                                                      (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 422 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Plasmid pSE1 "site binding to HindIII"                              fragment                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       AGCTGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCC60                 GGTGAGTCGC GTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTA120               GGTAGGCTCCAAGGGAGCCGGACAAAGGCCCGGTCTCGACCTGAGCTCTAAACTTACCTA180                GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTT TGTTT240               CGTTTTCTGTTCTGCGCCGTTACAACTTCAAGGTATGCGCTGGGACCTGGCAGGCGGCAT300                CTGGGACCCCTAGGAAGGGCTTGGGGGTCCTCGTGCCCAAGGCAGGGAACATAGTGGTCC360                CAGGAAGGGGAGCAGAGGCATCAGGGTGTCCA CTTTGTCTCCGCAGCTCCTGAGCCTGCA420               GA422                                                                          (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 77 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                        (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: Synthetic HindIII-"site binding to BamHI"                           fragment                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       AGCTTGTCGACTAATACGACTCACTATAGGGCGGCCGCGGGCCCCTGCAGGAATTCGGAT60                 CCCCCGGGTGACTGA CT77                                                           (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 61 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Synthetic HindIII-AccI fragment                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       AGCTTGCCGCCACTATGTCCGCAGTAAAAGCAGCCCGCTACGGCAAGGACAATGTCCGCG60                 T61                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                              (i ) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 920 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: HindIII-SnaBI fragment                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       AGCTTGCCGCCACTATGTCCGCAGTAAAAGCAGCCCGCTACGGC AAGGACAATGTCCGCG60                TCTACAAGGTTCACAAGGACGAGAAGACCGGTGTCCAGACGGTGTACGAGATGACCGTCT120                GTGTGCTTCTGGAGGGTGAGATTGAGACCTCTTACACCAAGGCCGACAACAGCGTCATTG180                TCGCAACCGACTCCATTAAGA ACACCATTTACATCACCGCCAAGCAGAACCCCGTTACTC240               CTCCCGAGCTGTTCGGCTCCATCCTGGGCACACACTTCATTGAGAAGTACAACCACATCC300                ATGCCGCTCACGTCAACATTGTCTGCCACCGCTGGACCCGGATGGACATTGACGGCAAGC360                CACACCCTCACTCCTTCATCCGCGACAGCGAGGAGAAGCGGAATGTGCAGGTGGACGTGG420                TCGAGGGCAAGGGCATCGATATCAAGTCGTCTCTGTCCGGCCTGACCGTGCTGAAGAGCA480                CCAACTCGCAGTTCTGGGGCTTCCTGCGTGACGAGTACACCACA CTTAAGGAGACCTGGG540               ACCGTATCCTGAGCACCGACGTCGATGCCACTTGGCAGTGGAAGAATTTCAGTGGACTCC600                AGGAGGTCCGCTCGCACGTGCCTAAGTTCGATGCTACCTGGGCCACTGCTCGCGAGGTCA660                CTCTGAAGACTTTTGCTGAAG ATAACAGTGCCAGCGTGCAGGCCACTATGTACAAGATGG720               CAGAGCAAATCCTGGCGCGCCAGCAGCTGATCGAGACTGTCGAGTACTCGTTGCCTAACA780                AGCACTATTTCGAAATCGACCTGAGCTGGCACAAGGGCCTCCAAAACACCGGCAAGAACG840                CCGAGGTCTTCGCTCCTCAGTCGGACCCCAACGGTCTGATCAAGTGTACCGTCGGCCGGT900                CCTCTCTGAAGTCTAAATTG920                                                    

What is claimed is:
 1. A protein possessing a specific urate oxidase activity of at least 16 U/mg and having the following sequence (SEQ ID NO:1):

    ______________________________________                                         Ser  Ala    Val    Lys  Ala  Ala  Arg  Tyr  Gly  Lys                           Asp  Asn    Val    Arg  Val  Tyr  Lys  Val  His  Lys                           Asp  Glu    Lys    Thr  Gly  Val  Gln  Thr  Val  Tyr                           Glu  Met    Thr    Val  Cys  Val  Leu  Leu  Glu  Gly                           Glu  Ile    Glu    Thr  Ser  Tyr  Thr  Lys  Ala  Asp                           Asn  Ser    Val    Ile  Val  Ala  Thr  Asp  Ser  Ile                           Lys  Asn    Thr    Ile  Tyr  Ile  Thr  Ala  Lys  Gln                           Asn  Pro    Val    Thr  Pro  Pro  Glu  Leu  Phe  Gly                           Ser  Ile    Leu    Gly  Thr  His  Phe  Ile  Glu  Lys                           Tyr  Asn    His    Ile  His  Ala  Ala  His  Val  Asn                           Ile  Val    Cys    His  Arg  Trp  Thr  Arg  Met  Asp                           Ile  Asp    Gly    Lys  Pro  His  Pro  His  Ser  Phe                           Ile  Arg    Asp    Ser  Glu  Glu  Lys  Arg  Asn  Val                           Gln  Val    Asp    Val  Val  Glu  Gly  Lys  Gly  Ile                           Asp  Ile    Lys    Ser  Ser  Leu  Ser  Gly  Leu  Thr                           Val  Leu    Lys    Ser  Thr  Asn  Ser  Gln  Phe  Trp                           Gly  Phe    Leu    Arg  Asp  Glu  Tyr  Thr  Thr  Leu                           Lys  Glu    Thr    Trp  Asp  Arg  Ile  Leu  Ser  Thr                           Asp  Val    Asp    Ala  Thr  Trp  Gln  Trp  Lys  Asn                           Phe  Ser    Gly    Leu  Gln  Glu  Val  Arg  Ser  His                           Val  Pro    Lys    Phe  Asp  Ala  Thr  Trp  Ala  Thr                           Ala  Arg    Glu    Val  Thr  Leu  Lys  Thr  Phe  Ala                           Glu  Asp    Asn    Ser  Ala  Ser  Val  Gln  Ala  Thr                           Met  Tyr    Lys    Met  Ala  Glu  Gln  Ile  Leu  Ala                           Arg  Gln    Gln    Leu  Ile  Glu  Thr  Val  Glu  Tyr                           Ser  Leu    Pro    Asn  Lys  His  Tyr  Phe  Glu  Ile                           Asp  Leu    Ser    Trp  His  Lys  Gly  Leu  Gln  Asn                           Thr  Gly    Lys    Asn  Ala  Glu  Val  Phe  Ala  Pro                           Gln  Ser    Asp    Pro  Asn  Gly  Leu  Ile  Lys  Cys                           Thr  Val    Gly    Arg  Ser  Ser  Leu  Lys  Ser  Lys                           Leu                                                                            ______________________________________                                    

preceded, if appropriate, by a methionine.
 2. A protein according to claim 1, wherein said protein is produced by recombinant methods.
 3. A protein according to claim 1, which represents, by analysis on a bidimensional Laemmli/SDS-Agarose gel, a spot of molecular mass of about 33.5 kDa, representing at least 90% of the protein mass.
 4. A protein according to claim 1, having a purity degree, determined by liquid chromatography on a C8 grafted silica column, higher than 80%.
 5. A protein according to claim 1, having an isoelectric point around 8.0.
 6. A protein according to claim 1, which carries a blocking group on the amino-terminal serine.
 7. A pharmaceutical composition comprising a protein according to claim
 1. 8. A protein according to claim 6, wherein said blocking group is an acetyl group.
 9. A protein according to claim 2, possessing a specific urate oxidase activity of about 30 U/mg. 